Nucleic Acid Amplification

ABSTRACT

Methods and compositions for the amplification of nucleic acids and generation of concatemers are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as nucleic acid polymerases and primers.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 5, 2015, is named 2026_502_SL.txt and is 36,588 bytes in size.

BACKGROUND

There is an increasing need for methods and reagents for the amplification of nucleic acids. Generation of multiple copies of a particular nucleic acid is often necessary or helpful in order for the nucleic acid to be used for a given application. For example, in order to analyze the nucleotide sequence of a nucleic acid of interest, frequently, the nucleic acid is replicated to increase its copy number before the sequence is analyzed. In another example, in order to determine the presence or absence of a particular nucleic acid in a sample, a sample may be treated under conditions such that if the particular nucleic acid is present in the sample, it may be amplified. In another example, a nucleic acid for use as a probe may be copied repeatedly to generate a large number of nucleic acids containing the same sequence as the original nucleic acid template, thereby generating many copies of the nucleic acid which may be used as a probe.

A variety of methods for the amplification of nucleic acids are known. For example, polymerase chain reaction (“PCR”) (see, e.g. U.S. Pat. No. 4,683,202) is a popular method for the amplification of nucleic acids. To successfully perform a PCR reaction, the reaction must be performed at multiple different temperatures, which are repeated for multiple cycles. This requires hardware or other mechanisms for repeatedly changing the temperature of the PCR reaction. Another method for amplification of nucleic acids is referred to as loop-mediated isothermal amplification (“LAMP”) (see, e.g. U.S. Pat. No. 6,410,278). LAMP reactions may be performed isothermally, but typically involve the use of four different primers which recognize a total of six distinct sequences on the target nucleic acid.

To facilitate the generation of amplified nucleic acids for the many and growing number of applications which use amplified nucleic acids, new methods and reagents for the amplification of nucleic acids are desired.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, in the event of a conflict between the content of the present express disclosure and the content of a document incorporated by reference herein, the content of the present express disclosure controls.

SUMMARY

Provided herein are methods and compositions relating to the amplification of nucleic acids and the generation of concatemers.

In some embodiments, provided herein is a method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising: (A) treating a primary double-stranded nucleic acid comprising the double-stranded nucleic acid template with a first copy of a first primer and a polymerase under conditions such that an extension product of the first copy of the first primer is synthesized which is annealed to a first strand of the double-stranded nucleic acid template, wherein the first primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising: (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and the template-binding region of the first copy of the first primer anneals to the first strand of the double-stranded nucleic acid template, (B) treating the extension product of the first copy of the first primer of step (A) with a second primer and a polymerase under conditions such that an extension product of the second primer is synthesized which is annealed to the extension product of the first copy of the first primer of step (A), wherein the second primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer, the template-binding region of the second primer anneals to the extension product of the first copy of the first primer of step (A), and the extension product of the second primer contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region comprising the 3′ terminal nucleotide, wherein the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the second primer read in the 5′ to 3′ direction, and the final nucleotide of the 3′ terminal region is the 3′ terminal nucleotide of the extension product of the second primer, (C) treating the extension product of the second primer of step (B) with a second copy of the first primer and a polymerase under conditions such that an extension product of the second copy of the first primer is synthesized which is annealed to the extension product of the second primer of step (B), to produce a first copy of a secondary nucleic acid comprising the extension product of the second primer of step (B) and the extension product of the second copy of the first primer, wherein the extension product of the second copy of the first primer contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region comprising the 3′ terminal nucleotide, wherein the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the first primer read in the 5′ to 3′ direction, and the final nucleotide of the 3′ terminal region is the 3′ terminal nucleotide of the extension product of the second primer, (D) repeating at least step (C) one or more addition times to generate at least a second copy of the secondary nucleic acid of step (C), (E) treating the first copy of the secondary nucleic acid of step (C) and the second copy of the secondary nucleic acid of step (D) under conditions such that the 3′ terminal region of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid anneals to the 3′ terminal region of the extension product of the second primer of the second copy of the secondary nucleic acid, to produce a cross-over structure comprising the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the second primer of the second copy of the secondary nucleic acid, (F) treating the cross-over structure of step (E) with a polymerase under conditions such that an extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid is synthesized and an extension product of the extension product of the second primer of the second copy of the secondary nucleic acid is synthesized, to produce a concatemer comprising two copies of the double-stranded nucleic acid template of step (A), wherein the concatemer comprises the extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the extension product of the second primer of the second copy of the secondary nucleic acid.

In some embodiments, provided herein is a method for generating a concatemer comprising two or more copies of a polynucleotide template or an analogous sequence thereof, the method comprising, (A) treating a primary nucleic acid comprising the polynucleotide template with a first copy of a first primer and a polymerase under conditions such that an extension product of the first copy of the first primer is synthesized which is annealed to the polynucleotide template, wherein the first primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer, (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and the template-binding region of the first copy of the first primer anneals to the polynucleotide template, (B) treating the extension product of the first copy of the first primer of step (A) with a second primer and a polymerase under conditions such that an extension product of the second primer is synthesized which is annealed to the extension product of the first copy of the first primer of step (A), wherein the second primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer, the template-binding region of the second primer anneals to the extension product of the first copy of the first primer of step (A), and the extension product of the second primer contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region comprising the 3′ terminal nucleotide, wherein the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the second primer read in the 5′ to 3′ direction, and the final nucleotide of the 3′ terminal region is the 3′ terminal nucleotide of the extension product of the second primer, (C) treating the extension product of the second primer of step (B) with a second copy of the first primer and a polymerase under conditions such that an extension product of the second copy of the first primer is synthesized which is annealed to the extension product of the second primer of step (B), to produce a first copy of a secondary nucleic acid comprising the extension product of the second primer of step (B) and the extension product of the second copy of the first primer, wherein the extension product of the second copy of the first primer contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region comprising the 3′ terminal nucleotide, wherein the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the first primer read in the 5′ to 3′ direction, and the final nucleotide of the 3′ terminal region is the 3′ terminal nucleotide of the extension product of the second primer, (D) repeating at least step (C) one or more additional times to generate at least a second copy of the secondary nucleic acid comprising the extension product of the second primer of step (B) and the extension product of the second copy of the first primer of step (C), (E) treating the first copy of the secondary nucleic acid of step (C) and the second copy of the secondary nucleic acid of step (D) under conditions such that the 3′ terminal region of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid anneals to the 3′ terminal region of the extension product of the second primer of the second copy of the secondary nucleic acid, to produce a cross-over structure comprising the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the second primer of the second copy of the secondary nucleic acid, (F) treating the cross-over structure of step (E) with a polymerase under conditions such that an extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid is synthesized and an extension product of the extension product of the second primer of the second copy of the secondary nucleic acid is synthesized, to produce a concatemer comprising two copies of the polynucleotide template of step (A), wherein the concatemer comprises the extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the extension product of the second primer of the second copy of the secondary nucleic acid. In some embodiments, the nucleic acid polymerase of step (A) is a DNA polymerase. In some embodiments, the nucleic acid polymerase of step (A) is a reverse transcriptase.

In some embodiments, provided herein is a method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising, (A) preparing a reaction mixture comprising: (i) a primary nucleic acid comprising the double-stranded nucleic acid template (ii) an isolated nucleic acid polymerase, (iii) a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (a) a tail region comprising (1) the 5′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (b) a template-binding region comprising (1) the 3′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, wherein the template-binding region is complementary to a first strand of the nucleic acid template, (iv) a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (a) a tail region comprising (1) the 5′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (b) a template-binding region comprising (1) the 3′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and wherein the template-binding region is complementary to a second strand of the nucleic acid template, and wherein the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer, and (B) incubating the reaction mixture for at least 3 minutes without thermocycling.

In some embodiments, provided herein is a method for generating a concatemer comprising two or more copies of a polynucleotide template, the method comprising, (A) preparing a reaction mixture comprising: (i) a nucleic acid comprising the polynucleotide template (ii) an isolated nucleic acid polymerase, (iii) a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (a) a tail region comprising (1) the 5′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (b) a template-binding region comprising (1) the 3′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and wherein the template-binding region is complementary to the polynucleotide template, (iv) a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (a) a tail region comprising (1) the 5′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (b) a template-binding region comprising (1) the 3′ terminal nucleotide of the primer (2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and wherein the template-binding region is complementary to a nucleotide sequence complementary to the polynucleotide template, and wherein the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences of the primers are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer, and (B) incubating the reaction mixture at a temperature of no greater than 80 C for at least 3 minutes.

In some embodiments, provided herein is a method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising incubating together a first copy and a second copy of a double-stranded nucleic acid molecule comprising the double-stranded nucleic acid template and a polymerase, wherein the double-stranded nucleic acid molecule comprises a first strand and a second strand, each containing a plurality of nucleotides, the first strand comprises a 5′ terminal nucleotide and a 3′ terminal nucleotide and contains the general format of regions in the 5′ to 3′ direction: A1-B-A2, the second strand comprises a 5′ terminal nucleotide and a 3′ terminal nucleotide and contains the general format of regions in the 5′ to 3′ direction: C1-D-C2, region B comprises the nucleotide sequence a first strand of the double-stranded nucleic acid template, region D comprises the nucleotide sequence of a second strand of the double-stranded nucleic acid template, in the double-stranded nucleic acid molecule, region A1 is annealed to C2, B is annealed to D, and A2 is annealed to C1, a cross-over structure comprising the first strand of the first copy of the double-stranded nucleic acid molecule and the second strand of the second copy of the double-stranded nucleic acid molecule is generated, wherein the A2 region of the first strand is annealed to the C2 region of the second strand, an extension product of the first strand of the cross-over structure is synthesized and an extension product of the second strand of the cross-over structure is synthesized, to produce a concatemer comprising the extension product of the first strand of the cross-over structure annealed to the extension product of the second strand of the cross-over structure, wherein the concatemer contains two copies of the double-stranded nucleic acid template.

In embodiments, provided herein is a method of copying a polynucleotide template, the method comprising: incubating the polynucleotide template in a reaction mixture comprising multiple copies of a first primer and multiple copies of a second primer, wherein: the first primer comprises a first region and a second region, wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and upon incubation of the polynucleotide template with the multiple copies of the first primer and the multiple copies of the second primer, at least one concatemer strand is formed, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides.

In some embodiments, in a method provided herein involving the formation of a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, the concatemer strand is a first concatemer strand, and a second concatemer strand is also formed, wherein the second concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C-X′-T′-C-T′-C, wherein: C represents the nucleotide sequence of the first region of the first primer, T′ represents a nucleotide sequence which is complementary to the polynucleotide template, and X′ represents a nucleotide sequence which is complementary to the nucleotide sequence of X.

In embodiments, provided herein is method of assaying for a target polynucleotide template in a biological sample, the method comprising: A) incubating the biological sample or portion thereof in a reaction mixture comprising multiple copies of a first primer and multiple copies of a second primer, wherein: the first primer comprises a first region and a second region, wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and upon incubation of the polynucleotide template with the multiple copies of the first primer and the multiple copies of the second primer, at least one concatemer strand is formed, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides; and B) measuring an amount of amplified nucleic acid in the reaction mixture of A) at one or more points after the initiation of the incubating step of A). In embodiments, the measuring an amount of amplified nucleic acid in the reaction mixture may comprise determining a level of fluorescence in the reaction mixture. In embodiments, the method may further comprise determining an inflection time of nucleic acid amplification in the reaction mixture.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, X may contain a sequence having the general structure in the 5′ to 3′ direction of [(C′-T)_(N)] wherein C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and N is any integer between 0 and 2000. In embodiments, N may be any integer between 0 and 10, 0 and 100, 0 and 1000, 0 and 5000, 0 and 10,000 1 and 10, 1 and 100, 1 and 1000, 1 and 2000, 1 and 5000, or 1 and 10,000.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, X may contain no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1000, 10,000, 50,000, 100,000, or 500,000 nucleotides. In embodiments, in a method, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, X may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1000, 10,000, 50,000, 100,000, or 500,000 nucleotides. In embodiments, in a method, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, X may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1000, 10,000, 50,000, 100,000, nucleotides, and no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1000, 10,000, 50,000, 100,000, or 500,000 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, between at least one C′ and T, one or more extra nucleotides are present which are not part of the C′ or T sequence. The one or more extra nucleotides may be, for example, between 1 and 10, 1 and 20, 1 and 100, or 1 and 1000 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, at least one C′ or T sequence may be missing one or more nucleotides. In the event that 2 or more nucleotides are missing, the missing nucleotides may be contiguous, or may be at separate locations. The one or more missing nucleotides may be, for example, between 1 and 10, 1 and 20, 1 and 100, or 1 and 1000 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, at least one C′ or T sequence may have one or more point mutations. In the event that two or more point mutations are present, the point mutations may be contiguous, or may be at separate locations. The one or more point mutations may be, for example, between 1 and 10, 1 and 20, 1 and 100, or 1 and 1000 point mutations.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, the nucleotide sequence has two or all three of the following characteristics: i) between at least one C′ and T, one or more extra nucleotides are present which are not part of the C′ or T sequence; ii) at least one C′ or T sequence is missing one or more nucleotides; and iii) at least one C′ or T sequence contains one or more point mutations.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, in embodiments which the polynucleotide template is an RNA molecule, the T may represent the nucleotide sequence of a DNA sequence which is analogous to the RNA sequence of the polynucleotide template.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, the concatemer strand further comprises one or more nucleotides to the 5′ of the 5′-most situated C′ sequence. The one or more nucleotides may be, for example, between 1 and 10, 1 and 20, 1 and 100, or 1 and 1000 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, the concatemer strand further comprises one or more nucleotides to the 3′ of the 3′-most situated C′ sequence. The one or more nucleotides may be, for example, between 1 and 10, 1 and 20, 1 and 100, or 1 and 1000 nucleotides.

In embodiments, provided herein is a method of generating a concatemer comprising at least two copies of a double stranded nucleic acid template, the method comprising: incubating in a reaction mixture at least a first template molecule and a second template molecule, wherein: the first template molecule comprises a first nucleic acid strand and a second nucleic acid strand, wherein: the first nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₁-H′, wherein: H′ represents the nucleotide sequence of a first homology sequence, S represents the nucleotide sequence of a first strand of the double stranded nucleic acid template, and Y₁ represents any number and sequence of nucleotides; and the second nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H-Y₁′-S′-H, wherein: H represents the nucleotide sequence of a second homology sequence, wherein the first homology sequence and second homology sequence are complementary to each other, Y₁′ represents a nucleotide sequence which is complementary to the nucleotide sequence of Y₁, and S′ represents the nucleotide sequence of a second strand of the double stranded nucleic acid template, wherein the first strand and second strand of the double stranded nucleic acid template are complementary to each other; and the second template molecule comprises a first nucleic acid strand and a second nucleic acid strand, wherein: the first nucleic acid strand of the second template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₂-H′, wherein: H′ represents the nucleotide sequence of the first homology sequence, S represents the nucleotide sequence of the first strand of the double stranded nucleic acid template, and Y₂ represents any number and sequence of nucleotides; and the second nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H-Y₂′-S′-H, wherein: H represents the nucleotide sequence of the second homology sequence, Y₂′ represents a nucleotide sequence which is complementary to the nucleotide sequence of Y₂, and S′ represents the nucleotide sequence of the second strand of the double stranded nucleic acid template; and upon incubation of the first template molecule with the second template molecule in the reaction mixture, at least one concatemer comprising at least two copies of the double stranded nucleic acid template is formed, wherein the concatemer comprises a first concatemer strand and a second concatemer strand, wherein the first concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₂-H′-S-Y₁-H′, wherein each of H′, Y₁, S, and Y₂ represent nucleotide sequences as described above; and wherein the second concatemer strand comprises a 5′ end and a 3′ end, and comprises a sequence having the general structure in the 5′ to 3′ direction of: H-Y₁′-S′-H-Y₂′-S′-H, wherein each of H′, Y₁, S, and Y₂ represent nucleotide sequences as described above.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₂-H′-S-Y₁-H′, at least one of or both Y₁ and Y₂ may represent 0 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₂-H′-S-Y₁-H′, Y₁ may contain a sequence having the general structure in the 5′ to 3′ direction of [(H′-S)_(N1)] wherein H′ and S represent nucleotide sequences as described above, and N1 is any integer between 0 and 2000.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first concatemer strand which comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₂-H′-S-Y₁-H′, Y₂ may contain a sequence having the general structure in the 5′ to 3′ direction of [(H′-S)_(N2)] wherein H′ and S represent nucleotide sequences as described above, and N2 is any integer between 0 and 2000.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first template molecule and a second template molecule, the first template molecule and second template molecule are both double-stranded DNA molecules.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first nucleic acid strand of a first template molecule comprises, wherein the first nucleic acid strand comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y₁-H′, wherein H′ represents the nucleotide sequence of a first homology sequence, the first homology sequence may contain no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, the first homology sequence may contain at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, the first homology sequence may contain at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 and no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or 200 nucleotides.

In embodiments, a reaction mixture, vessel, or kit provided herein comprises a nucleic acid polymerase. In embodiments, a nucleic acid polymerase is a DNA polymerase having strand-displacement activity. In embodiments, a nucleic acid polymerase is an RNA polymerase. In embodiments, a nucleic acid polymerase is a reverse transcriptase. In embodiments, a reaction mixture, vessel, or kit comprises more than one kind of nucleic acid polymerase, such as both a DNA polymerase having strand displacement activity and a reverse transcriptase. In embodiments, a reaction mixture, vessel, or kit provided herein comprises nucleotides and buffer.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template is a single-stranded molecule. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template comprises one strand of a double-stranded nucleic acid template. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template is a DNA or RNA molecule.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a nucleic acid template, the nucleic acid template is an RNA or DNA molecule. In embodiments, a nucleic acid template may be a single-stranded or double-stranded molecule.

In embodiments, in a method provided herein involving incubation of a reaction mixture, during the incubation of the reaction mixture, the temperature of the reaction mixture does not exceed 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 37, 35, 30, 25, or 20 C. In embodiments, in a method provided herein, all steps of the method are performed at a temperature of no greater than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 37, 35, 30, 25, or 20 C. In embodiments, a reaction mixture, vessel, or kit provided herein is maintained at a temperature of no greater than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 37, 35, 30, 25, or 20 C. In embodiments, a method provided herein is performed without thermocycling.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template comprising a first portion, the first portion contains no more than 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 200 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template comprising a first portion, the first portion contains at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 200 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template comprising a first portion, the first portion contains at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 and no more than 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 200 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a first region, the first region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a first region, the first region contains no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a first region, the first region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90 and no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a first region and a second primer comprising a first region, both the first primer and second primer may have any of the features described above. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a first region and a second primer comprising a first region, the first region of the first primer and the first region of the second primer may contain the same number of nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a first region and a second primer comprising a first region, the first region of the first primer and the first region of the second primer may contain a different number of nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a second region, the second region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a second region, the second region contains no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a second region, the second region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90 and no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a second region and a second primer comprising a second region, both the first primer and second primer may have any of the features described above. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a second region and a second primer comprising a second region, the second region of the first primer and the second region of the second primer may contain the same number of nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a second region and a second primer comprising a second region, the second region of the first primer and the second region of the second primer may contain a different number of nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a second primer containing a second region and a polynucleotide template comprising a second portion, the nucleotide sequence of the second region of the second primer is the same as the nucleotide sequence of the second portion of the polynucleotide template.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a tail region, the tail region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a tail region, the tail region contains no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a tail region, the tail region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90 and no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a tail region and a second primer comprising a tail region, both the first primer and second primer may have any of the features described above. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a tail region and a second primer comprising a tail region, the tail region of the first primer and the tail region of the second primer may contain the same number of nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a tail region and a second primer comprising a tail region, the tail region of the first primer and the tail region of the second primer may contain a different number of nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a template-binding region, the template-binding region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a template-binding region, the template-binding region contains no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a primer comprising a template-binding region, the template-binding region contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90 and no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a template-binding region and a second primer comprising a template-binding region, both the first primer and second primer may have any of the features described above. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a template-binding region and a second primer comprising a template-binding region, the template-binding region of the first primer and the template-binding region of the second primer may contain the same number of nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a first primer comprising a template-binding region and a second primer comprising a template-binding region, the template-binding region of the first primer and the template-binding region of the second primer may contain a different number of nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or 5000 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template may contain no more than 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, or 10,000 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a polynucleotide template, the polynucleotide template may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or 5000, and no more than 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, or 10,000 nucleotides.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a double-stranded nucleic acid template, each strand of the double-stranded nucleic acid template may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or 5000 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a double-stranded nucleic acid template, each strand of the double-stranded nucleic acid template may contain no more than 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, or 10,000 nucleotides. In embodiments, in a method, reaction mixture, vessel, or kit provided herein involving a double-stranded nucleic acid template, each strand of the double-stranded nucleic acid template may contain at least 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or 5000, and no more than 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, or 10,000 nucleotides.

In embodiments, a reaction mixture, vessel, or kit provided herein does not contain a recombinase enzyme.

In embodiments, in a method, reaction mixture, vessel, or kit provided herein may contain or involve multiple copies of a primer. The multiple copies may be, for example, at least 5, 10, 15, 20, 50, 100, 500, 1000, 10,000, 100,000, or 1,000,000 copies of the primer.

In embodiments, a reaction mixture or vessel provided herein may comprise at least a portion of a biological sample from a subject. The biological sample may be, for example, saliva, blood, urine, a cheek swab, or a nasal swab. The subject may be a human.

In some embodiments, all of the steps of a method provided herein are performed at a temperature of no greater than 70, 65, 60, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 C. In some embodiments, some of the steps of a method provided herein are performed at a temperature of no greater than 70, 65, 60, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 C.

In some embodiments, two or more steps of a method provided herein are performed simultaneously in the same reaction mixture. In some embodiments, all of the steps of a method provided herein are performed simultaneously in the same reaction mixture.

In some embodiments, in a method provided herein, a nucleic acid template is amplified at least 10, 100, 1000, 10,000, 100,000, or 1,000,000-fold within 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 120, or 180 minutes of initiation of the method. In some embodiments, in a method provided herein, the number of copies of a nucleic acid template in a reaction mixture is increased least 10, 100, 1000, 10,000, 100,000, or 1,000,000-fold within 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 120, or 180 minutes of initiation of the method.

In embodiments, a nucleic acid template provided herein may be a single-stranded or a double-stranded nucleic acid template.

In embodiments, provided herein is a vessel, comprising in fluid communication therein: a first primer, wherein the first primer comprises a first region and a second region, and wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of a polynucleotide template; a second primer, wherein the second primer comprises a first region and a second region, and wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and at least one concatemer strand, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides.

In some embodiments, provided herein is a vessel, comprising in fluid communication therein: (A) an isolated nucleic acid polymerase, (B) a nucleic acid template comprising at least a first strand, (C) a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, wherein the template-binding region is complementary to a first strand of the nucleic acid template, and (D) a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and wherein the template-binding region is complementary to a nucleotide sequence complementary to first strand of the nucleic acid template, and wherein the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences of the primers are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer.

In embodiments, provided herein is a kit comprising two or more fluidically isolated containers, the containers collectively comprising: a first primer, wherein the first primer comprises a first region and a second region, and wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of a polynucleotide template; a second primer, wherein the second primer comprises a first region and a second region, and wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and an isolated DNA polymerase having strand-displacement activity; wherein: the first region of the first primer and the first region of the second primer are complementary.

In some embodiments, provided herein is a kit for detecting a target nucleic acid of interest comprising at least a first strand, the kit comprising two or more fluidically isolated containers, the containers collectively comprising: (A) an isolated nucleic acid polymerase, (B) a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, wherein the template-binding region is complementary to the first strand of the target nucleic acid, and (C) a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions: (i) a tail region comprising (a) the 5′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide (c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and (ii) a template-binding region comprising (a) the 3′ terminal nucleotide of the primer (b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide (c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and wherein the template-binding region is complementary to a nucleotide sequence complementary to the first strand of the target nucleic acid, and wherein the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences of the primers are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer.

In some embodiments, a kit provided herein comprises a nucleic acid having the nucleotide sequence of the target nucleic acid of interest.

In some embodiments, a reaction mixture, vessel or kit provided herein comprises a nucleic acid dye.

In some embodiments, in a vessel or kit provided herein comprising an isolated nucleic acid polymerase, the isolated nucleic acid polymerase is a DNA polymerase. In some embodiments, in a vessel or kit provided herein comprising an isolated nucleic acid polymerase, the isolated nucleic acid polymerase is a reverse transcriptase. In some embodiments, in a vessel or kit provided herein comprising an isolated nucleic acid polymerase, the vessel or kit comprises both a DNA polymerase and a reverse transcriptase.

In some embodiments, in a method, vessel, or kit provided herein comprising a nucleic acid polymerase, the nucleic acid polymerase has strand displacement activity.

In some embodiments, a method provided herein comprises treating one or more of the reaction components or steps of the method with a nucleic acid dye.

In some embodiments, in a method, vessel, or kit provided herein comprising a nucleic acid template, the template is a DNA molecule. In some embodiments, in a method, vessel, or kit provided herein comprising a nucleic acid template, the template is an RNA molecule.

In some embodiments, in a method, vessel, or kit provided herein comprising a first primer, the tail region of the first primer comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, in a method, vessel, or kit provided herein comprising a first primer, the tail region of the first primer comprises no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or 60 nucleotides.

In some embodiments, in a method, vessel, or kit provided herein comprising a second primer, the tail region of the second primer comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, in a method, vessel, or kit provided herein comprising a second primer, the tail region of the second primer comprises no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or 60 nucleotides.

In some embodiments, in a method, vessel, or kit provided herein comprising a first primer, the template-binding region of the first primer comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, in a method, vessel, or kit provided herein comprising a first primer, the template-binding region of the first primer comprises no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or 60 nucleotides.

In some embodiments, in a method, vessel, or kit provided herein comprising a second primer, the template-binding region of the second primer comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, in a method, vessel, or kit provided herein comprising a second primer, the template-binding region of the second primer comprises no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or 60 nucleotides.

In some embodiments, in methods and compositions provided herein wherein an RNA molecule is the template molecule or primary nucleic acid, amplification of the template may refer to generation of copies of DNA strands corresponding to the RNA molecule.

In some embodiments, a method provided herein comprises measuring a fluorescent signal from an assay comprising the method.

In some embodiments, a nucleic acid ligase may be included with a method or composition provided herein. In some embodiments, a nucleic acid template may be amplified more rapidly with a method provided herein when a ligase is included in a reaction mixture for a method provided herein, as compared to if a nucleic acid ligase is not included in the reaction. In embodiments, a reaction mixture, vessel, or kit provided herein may contain an enzyme having ligase activity.

In embodiments, provided herein is a method of assaying for a pathogen in a sample, the method comprising performing a method as provided herein to amplify nucleic acid from the pathogen. In embodiments, the target nucleic acid used in a composition or method provided herein may be nucleic acid from a pathogen. In embodiments, the first and second primer used in a method provided herein may each contain regions which are complementary to a sequence in the nucleic acid of the pathogen, or which are complementary to a sequence which is complementary to a sequence in the nucleic acid of the pathogen. In embodiments, the nucleic acid of the pathogen may be DNA or RNA. Pathogens may include, without limitation, viruses, bacteria, fungi, and protists. A sample may be from a subject, and may have any of the sample characteristics described elsewhere herein.

In embodiments, a method provided herein for amplification of a nucleic acid may be used for a diagnostic method externally of a human or animal body. For example, a sample may be obtained from a human or animal, and the sample may be assayed for a target nucleic acid of interest with a method provided herein for amplification of nucleic acid.

In embodiments, a method provided herein may include: a) providing one or more reagents for performing a method as provided herein (e.g. one or more of first primer, second primer, nucleic acid template, nucleic acid polymerase, nucleotides, buffer, water, etc.) in a reaction mixture, and b) incubating the reaction mixture at a substantially isothermal temperature, wherein the temperature of the reaction mixture does not diverge from a central temperature by more or less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 degree Celsius during the incubation. In embodiments, a central temperature may be, for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 degrees Celsius.

In embodiments, a method provided herein may be performed at a substantially isothermal temperature. In embodiments, a substantially isothermal temperature may be any of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 degrees Celsius, plus or minus 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 degree Celsius.

In embodiments, a method provided herein may be performed at one or more temperatures, none or which exceed 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25 degrees Celsius.

In embodiments, methods provided herein may be performed without thermocycling/reaction mixtures may be incubated under conditions without thermocycling (i.e. without cycles of raising and lowering incubation temperatures to separate strands or allow hybridization of primers as is used in, for example, PCR-based methods).

In compositions and methods provided herein involving a first primer comprising a first region and a second primer comprising a first region, wherein the first region of the first primer is complementary to the first region of the second primer, in embodiments, the first region of the first primer and the first region of the second primer contain nucleotide sequences such that a double stranded structure which would be formed by the annealing of the first region of the first primer to the first region of the second primer according to Watson-Crick base pairing rules would not form a restriction enzyme recognition sequence.

In compositions and methods provided herein involving a nucleic acid polymerase, in embodiments, the nucleic acid polymerase has 3′ to 5′ exonuclease activity.

In embodiments, provided herein is a method for amplifying a target nucleic acid strand, the method comprising: incubating a reaction mixture comprising the target nucleic acid strand, a first primer, and a second primer under substantially isothermal conditions, wherein: the target nucleic acid strand comprises a first portion and a second portion; the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the nucleic acid strand; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to the second portion of the nucleic acid strand; the first region of the first primer is complementary to the first region of the second primer; and the target nucleic acid strand is amplified. Optionally, the target nucleic acid strand may further comprise a third portion, wherein the third portion is situated in the target nucleic acid strand between the first portion and the second portion, and wherein the first region of the second primer is complementary to the third portion of the target nucleic acid strand.

In embodiments, in any of the methods provided herein, a, polynucleotide template, target nucleic acid strand or the like may contain an internal motif, wherein the tail region of a primer provided herein to amplify the target nucleic acid strand is complementary to the internal motif.

In embodiments, a method provided herein may further comprise adding to a reaction mixture provided herein a peptide-nucleic acid (PNA) probe and a dye which binds to DNA-PNA hybrid, wherein the PNA probe is complementary to a target nucleic acid for amplification in the reaction mixture, or a complement thereof.

In embodiments, a reaction mixture provided herein may comprise a DNA polymerase having strand-displacement activity.

When a nucleic acid is described herein as being “amplified” or the like, the nucleic acid may also be described as being “copied” or the like.

In embodiments, in a primer described herein as having a first region and a second region, the first region may contain the 5′ end of the primer and the second region may contain the 3′ end of the primer.

In embodiments, in a primer described herein as having a tail region and a template-binding region, the first region may contain the 5′ end of the primer and the second region may contain the 3′ end of the primer.

In embodiments, provided herein is method for copying a polynucleotide template, the method comprising: incubating a reaction mixture comprising the polynucleotide template, a first primer, and a second primer under conditions without thermocycling, wherein: the polynucleotide template comprises a first portion and a second portion; the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to the second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and multiple copies of the polynucleotide template are generated. Optionally, the polynucleotide template may further comprise a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion, and wherein the first region of the second primer is complementary to the third portion of the polynucleotide template.

In embodiments, provided herein is a method for amplifying a polynucleotide template, the method comprising incubating the polynucleotide template in a reaction mixture comprising a first primer and a second primer, wherein: the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion; the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template; and the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template.

In embodiments, provided herein is a method for assessing the identity of a nucleotide at a position of interest in a nucleotide sequence in a polynucleotide template, the method comprising: A) providing copies of the polynucleotide template in each of at least a first reaction mixture and a second reaction mixture, wherein: the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion and wherein the position of interest is in the third portion; the first reaction mixture comprises copies of the polynucleotide template, a first primer, and a second primer, wherein: the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and the first region of the second primer is complementary to the third portion of the polynucleotide template; the second reaction mixture comprises copies of the polynucleotide template, a third primer, and a fourth primer, wherein: the third primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of the polynucleotide template; the fourth primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the third primer is complementary to the first region of the fourth primer; and the first region of the fourth primer is complementary to the third portion of the polynucleotide template; and the nucleotide sequence of the first region of the second primer differs from the nucleotide sequence of first region of the fourth primer by a single nucleotide, wherein the position of the different nucleotide in the second and fourth primers corresponds to the position of the nucleotide of interest in the polynucleotide template if the nucleotide sequence of the first region of the second primer or fourth primer is oriented with the nucleotide sequence of the third portion of the polynucleotide template for maximum complementation of the sequences; B) incubating the first reaction mixture and second reaction mixture under conditions without thermocycling; and C) comparing the rate or amount of amplification of the polynucleotide template in the first reaction mixture to the rate or amount of amplification of the polynucleotide template in the second reaction mixture, wherein the rate or amount of amplification of the polynucleotide template is indicative of the degree of complementation between first region of the second or fourth primer and the nucleotide sequence of the third portion of the polynucleotide template.

Optionally, in embodiments provided herein involving a polynucleotide template, the polynucleotide template is a DNA strand.

Optionally, in embodiments provided herein involving a polynucleotide template, the polynucleotide template is an RNA strand.

Optionally, in embodiments provided herein involving a polynucleotide template, the polynucleotide template is one strand of a duplex nucleic acid molecule. In embodiments, the duplex nucleic acid molecule is a duplex DNA molecule or a duplex RNA molecule.

Optionally, in embodiments provided herein involving a reaction mixture, vessel, or kit, the reaction mixture, vessel, or kit comprises a DNA polymerase having strand displacement activity.

Optionally, in embodiments provided herein involving a reaction mixture, vessel, or kit, the reaction mixture, vessel, or kit comprises a reverse transcriptase.

Optionally, in embodiments provided herein involving a reaction mixture, vessel, or kit, the reaction mixture, vessel, or kit comprises a nucleic acid dye, nucleotides, or buffers.

Optionally, in embodiments provided herein involving a polynucleotide template containing a first portion, a second portion, and a third portion, the polynucleotide template may have the general structure of elements, in the 3′ to 5′ direction, of: 1P-1S-3P-2S-2P, wherein “1P” is the first portion, “1S” is a first space, “3P” is the third portion, “2S” is a second space, and “2P” is the second portion. The “first space” and “second space” are portions of the polynucleotide template which contain nucleotides which are not part of the first portion, second portion, or third portion. In embodiments, the first portion may have, for example, between 6 and 30 nucleotides or any other number of nucleotides as described elsewhere herein. In embodiments, the first space may, for example, between 2 and 30 nucleotides or any other number of nucleotides as described elsewhere herein. In embodiments, the third portion may have, for example, between 4 and 14 nucleotides or any other number of nucleotides as described elsewhere herein. In embodiments, the first space may, for example, between 2 and 30 nucleotides or any other number of nucleotides as described elsewhere herein. In embodiments, the first portion may have, for example, between 6 and 30 nucleotides or any other number of nucleotides as described elsewhere herein.

In embodiments, in methods provided herein involving the incubation of a polynucleotide template in a reaction mixture, a concatemer strand comprising at least 2, 3, 4, 5, or 10 copies of the polynucleotide template may be generated during the incubation of the reaction mixture.

In embodiments, provided herein is a method for amplifying a double stranded nucleic acid molecule, the method comprising incubating the double stranded nucleic acid molecule in a reaction mixture comprising a first primer and a second primer, wherein: the double stranded nucleic acid molecule comprises a first strand and a second strand, wherein the first strand comprises a first portion and a third portion and the second strand comprises a second portion; the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the first strand; and the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to the second portion of the second strand, the first region of the second primer is complementary to the third portion of the first strand, and the first region of the second primer is complementary to the first region of the first primer.

In embodiments, provided herein is a reaction mixture comprising: a polynucleotide template, a first primer, and a second primer, wherein: the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion; the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to the second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and the first region of the second primer is complementary to the third portion of the polynucleotide template.

In embodiments, provided herein is a kit for the amplification of a polynucleotide template, the kit comprising: a first primer and a second primer, wherein: the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and the first region of the second primer is complementary to a third portion of the polynucleotide template, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion.

Optionally, in embodiments provided herein involving a kit, the components of the kit may be distributed between at least two separate fluidically isolated containers.

In embodiments, provided herein is a kit comprising two or more fluidically isolated containers, the containers collectively comprising: a first primer, a second primer, a third primer, and a fourth primer, wherein: the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of a polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the third primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template; the fourth primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; the first region of the third primer is complementary to the first region of the fourth primer; and the nucleotide sequence of the first region of the first primer differs from the nucleotide sequence of first region of the third primer by a single nucleotide. Optionally, the first region of the second primer and the first region of the fourth primer are both complementary to a third portion of the polynucleotide template, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion. Optionally, the second region of the each of the first primer, second primer, third primer, and fourth primers is between 6 and 30 nucleotides in length. Optionally, the second region of the each of the first primer, second primer, third primer, and fourth primers is between 6 and 30 nucleotides in length.

In embodiments provided herein involving a kit, the kit may comprises a control nucleic acid strand comprising the nucleotide sequence of a polynucleotide template to be detected with reagents of the kit.

In methods and compositions provided herein, optionally, a PNA probe and the dye DiSc₂(5) may be included. The PNA probe may specifically anneal to a target sequence for amplification in the relevant reaction, and copies thereof, such that DNA-PNA duplexes are formed. Methods provided herein may include detecting a color change of the dye DiSc₂(5) upon the binding of the dye to DNA-PNA duplexes. In embodiments, a PNA probe and dye may be added to a reaction mixture provided herein after the initiation or after the completion of the reaction. In embodiments, a PNA probe and dye may be added to a reaction mixture provided herein at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 minutes after the initiation of the reaction. In embodiments, a PNA probe and dye may be added to a reaction mixture provided herein no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 minutes after the initiation of the reaction.

References herein to generating a copy of or amplifying a polynucleotide template or nucleic acid template include generating a copy which contains the sequence of the polynucleotide template/nucleic acid template, as well as generating a copy which contains an analogous sequence of the polynucleotide template/nucleic acid template, unless the context clearly dictates otherwise. For instance, if a polynucleotide template is RNA, generating a copy of the template can include generating a copy which is a DNA molecule which contains the DNA version of the RNA sequence of the polynucleotide template (i.e. in the DNA sequence, contains “T”s instead of “U”s).

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIGS. 1A-1K depict a general schematic of a method provided herein. FIGS. 1A-1K include the following nucleotide sequences: 103: AACGGTTGCTC (SEQ ID NO: 1); 104: GAGCAACCGTT (SEQ ID NO: 2); 105: ATGGGAGC (SEQ ID NO: 3); 106: CCATAACG (SEQ ID NO: 4); 111: ATGGGAGCAACCGTT (SEQ ID NO: 5); 112: CCATAACGGTTGCTCCCAT (SEQ ID NO: 6); 113: ATGGGAGCAACCGTTATGG (SEQ ID NO: 7); 121: CCATAACGGTTGCTCCCATAACGGTTGCTCCCAT (SEQ ID NO: 8); 122: ATGGGAGCAACCGTTATGGGAGCAACCGTTATGG (SEQ ID NO: 9).

FIG. 2A shows nucleotide sequences of certain primers used with methods provided herein. FIG. 2A includes the following nucleotide sequences: P1 primer, 37 nucleotides: CGCCGGATGGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 10); P1 primer, 35 nucleotides: CCGGATGGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 11); P1 primer, 33 nucleotides: GGATGGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 12); P1 primer, 31 nucleotides: ATGGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 13); P1 primer, 30 nucleotides: TGGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 14); P1 primer, 29 nucleotides: GGCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 15); P1 primer, 28 nucleotides: GCTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 16); P1 primer, 27 nucleotides: CTCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 17); P1 primer, 26 nucleotides: TCTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 18); P1 primer, 25 nucleotides: CTTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 19); P1 primer, 24 nucleotides: TTGGGAAACCAAACCGTACCAACC (SEQ ID NO: 20); P1 primer, 23 nucleotides: TGGGAAACCAAACCGTACCAACC (SEQ ID NO: 21); P2 primer, 36 nucleotides: GTTTCCCAAGAGCCATCCGGCGATGCGGAATGTACC (SEQ ID NO: 22); P2 primer, 34 nucleotides: GTTTCCCAAGAGCCATCCGGATGCGGAATGTACC (SEQ ID NO: 23); P2 primer, 32 nucleotides: GTTTCCCAAGAGCCATCCATGCGGAATGTACC (SEQ ID NO: 24); P2 primer, 30 nucleotides: GTTTCCCAAGAGCCATATGCGGAATGTACC (SEQ ID NO: 25); P2 primer, 29 nucleotides: GTTTCCCAAGAGCCAATGCGGAATGTACC (SEQ ID NO: 26); P2 primer, 28 nucleotides: GTTTCCCAAGAGCCATGCGGAATGTACC (SEQ ID NO: 27); P2 primer, 27 nucleotides: GTTTCCCAAGAGCATGCGGAATGTACC (SEQ ID NO: 28); P2 primer, 26 nucleotides: GTTTCCCAAGAGATGCGGAATGTACC (SEQ ID NO: 29); P2 primer, 25 nucleotides: GTTTCCCAAGAATGCGGAATGTACC (SEQ ID NO: 30); P2 primer, 24 nucleotides: GTTTCCCAAGATGCGGAATGTACC (SEQ ID NO: 31); P2 primer, 23 nucleotides: GTTTCCCAAATGCGGAATGTACC (SEQ ID NO: 32); and P2 primer, 22 nucleotides: GTTTCCCAATGCGGAATGTACC (SEQ ID NO: 33).

FIG. 2B is a graph depicting results from reactions performed according to a method provided herein.

FIGS. 3A and 3B show nucleotide sequences of certain primers used with methods provided herein. FIG. 3A includes the following nucleotide sequences: C5: ATGGCTCTTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 34); D5: GTTTCCCAAGAGCCATGGATGCGGAATGTACC (SEQ ID NO: 35); C6: GGCTCTTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 36); D6: GTTTCCCAAGAGCCGGATGCGGAATGTACC (SEQ ID NO: 37); C7: CTCTTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 38); D7: GTTTCCCAAGAGGGATGCGGAATGTACC (SEQ ID NO: 39); C8: TCTTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 40); D8: GTTTCCCAAGAGGATGCGGAATGTACC (SEQ ID NO: 41); C9: CTTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 42); D9: GTTTCCCAAGGGATGCGGAATGTACC (SEQ ID NO: 43); C10: TTGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 44); D10: GTTTCCCAAGGATGCGGAATGTACC (SEQ ID NO: 45); C11: TGGGAAACTGAAACCGTACCAACC (SEQ ID NO: 46); D11: GTTTCCCAGGATGCGGAATGTACC (SEQ ID NO: 47); C12: GGGAAACTGAAACCGTACCAACC (SEQ ID NO: 48); and D12: GTTTCCCGGATGCGGAATGTACC (SEQ ID NO: 49). FIG. 3B includes the following nucleotide sequences: A1: ATGGCTCTTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 50); B1: GTTTCCCAAGAGCCATACAGGGATGCGGAATGTACC (SEQ ID NO: 51); A2: GGCTCTTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 52); B2: GTTTCCCAAGAGCCACAGGGATGCGGAATGTACC (SEQ ID NO: 53); A3: CTCTTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 54); B3: GTTTCCCAAGAGACAGGGATGCGGAATGTACC (SEQ ID NO: 55); A4: TCTTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 56); B4: GTTTCCCAAGAACAGGGATGCGGAATGTACC (SEQ ID NO: 57); A5: CTTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 58); B5: GTTTCCCAAGACAGGGATGCGGAATGTACC (SEQ ID NO: 59); A6: TTGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 60); B6: GTTTCCCAAACAGGGATGCGGAATGTACC (SEQ ID NO: 61); A7: TGGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 62); B7: GTTTCCCAACAGGGATGCGGAATGTACC (SEQ ID NO: 63); A8: GGGAAACTGCCTGAAACCGTACCAACC (SEQ ID NO: 64); and B8: GTTTCCCACAGGGATGCGGAATGTACC (SEQ ID NO: 65).

FIGS. 3C and 3D are graphs depicting results from reactions performed according to a method provided herein.

FIG. 4 is a graph depicting results from reactions performed according to a method provided herein.

FIG. 5 is a graph depicting results from reactions performed according to a method provided herein.

FIG. 6 is a graph depicting results from reactions performed according to a method provided herein.

FIG. 7 is a sequence alignment depicting the nucleotide sequences of products generated in reactions performed according to a method provided herein. FIG. 7 includes the following nucleotide sequences (in order from the top sequence in the alignment to the bottom sequence in the alignment): SEQ ID NO: 66: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGA; SEQ ID NO: 67: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 68: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 69: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 70: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACTGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 71: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 72: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 73: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 74: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 75: TCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAA GACCAATTCTTGAGAGAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 76: GAACCCACTAACAGTAGAAGTACCATACATTTGTACAGAAGGGGAAGACCAAAT GAACCCACTAACAGTAGAAGTACCATACATTT; SEQ ID NO: 77: TCTTGAGAGAACCCACTAACTCTTGAGAGAACCCACTAAC; and SEQ ID NO: 78: GGAAGACCAAATTCTTGAGA.

FIGS. 8A-8C are general schematics of features of embodiments of molecules provided herein.

FIG. 9 is a general schematic of features of embodiments of molecules provided herein.

FIGS. 10A-10C are general schematics of features of embodiments of molecules provided herein.

FIG. 11 is a graph depicting results from reactions performed according to a method provided herein.

FIG. 12 is a graph depicting results from reactions performed according to a method provided herein.

It is noted that the drawings and elements therein are not necessarily drawn to shape or scale. For example, the shape or scale of elements of the drawings may be simplified or modified for ease or clarity of presentation. It should further be understood that the drawings and elements therein are for exemplary illustrative purposes only, and not be construed as limiting in any way.

DETAILED DESCRIPTION

While the invention includes various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.

In some embodiments, provided herein are methods and compositions relating to the amplification of nucleic acids and the generation of concatemers.

In methods provided herein, generation of nucleic acid concatemers also amplifies the number of copies of the nucleic acid template/particular nucleic acid in the concatemer. Accordingly, references herein to methods and compositions for the generation of concatemers also apply to the amplification of nucleic acids.

As used herein, a “polynucleotide” refers to a polymeric chain containing two or more nucleotides. “Polynucleotides” includes primers, oligonucleotides, nucleic acid strands, etc. A polynucleotide may contain standard or non-standard nucleotides. Typically, a polynucleotide contains a 5′ phosphate at one terminus (“5′ terminus”) and a 3′ hydroxyl group at the other terminus (“3′ terminus) of the chain. The most 5′ nucleotide of a polynucleotide may be referred to herein as the “5′ terminal nucleotide” of the polynucleotide. The most 3′ nucleotide of a polynucleotide may be referred to herein as the “3′ terminal nucleotide” of the polynucleotide.

The term “downstream” as used herein in the context of a polynucleotide containing a 5′ terminal nucleotide and a 3′ terminal nucleotide refers to a position in the polynucleotide which is closer to the 3′ terminal nucleotide than a reference position in the polynucleotide. For example, in a primer having the sequence: 5′ ATAAGC 3′, the “G” is downstream from the “T” and all of the “A”s.

The term “upstream” as used herein in the context of a polynucleotide containing a 5′ terminal nucleotide and a 3′ terminal nucleotide, refers to a position in the polynucleotide which is closer to the 5′ terminal nucleotide than a reference position in the polynucleotide. For example, in a primer having the sequence: 5′ ATAAGC 3′, the “T” is upstream from the “G”, the “C”, and the two “A”s closest to the “G”.

As used herein, “nucleic acid” includes both DNA and RNA, including DNA and RNA containing non-standard nucleotides. A “nucleic acid” contains at least one polynucleotide (a “nucleic acid strand”). A “nucleic acid” may be single-stranded or double-stranded.

As used herein, a “concatemer” refers to a nucleic acid molecule which contains within it two or more copies of a particular nucleic acid, wherein the copies are linked in series. Within the concatemer, the copies of the particular nucleic acid may be linked directly to each other, or they may be indirectly linked (e.g. there may be nucleotides between the copies of the particular nucleic acid). In an example, the particular nucleic acid may be that of a double-stranded nucleic acid template, such that a concatemer may contain two or more copies of the double-stranded nucleic acid template. In another example, the particular nucleic acid may be that of a polynucleotide template, such that a concatemer may contain two or more copies of the polynucleotide template.

As used herein, a “target” nucleic acid or molecule refers to a nucleic acid of interest. A target nucleic acid/molecule may be of any type, including single-stranded or double stranded DNA or RNA (e.g. mRNA).

As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which can pair with each other according to standard base-pairing rules (e.g. A-T, G-C, or A-U), such that molecules containing the sequences can specifically anneal to each other. It is not necessary for every nucleotide base in two sequences to be capable of pairing with each other for the sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences can pair with each other when the sequences are optimally aligned for complementation. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer can pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Additionally, “complementary” sequences may contain one or more nucleotide analogs or nucleobase analogs. As used herein, “perfectly complementary” or “perfect complementation” or the like refers two sequences which are 100% complementary to each other (i.e. where there are no mis-matches between the nucleotides of the sequences when the sequences are paired for maximum complementation).

As used herein, the term “isolated” as applied to proteins, nucleic acids, or other biomolecules refers to a molecule that has been purified or separated from a component of its naturally-occurring environment (e.g. a protein purified from a cell in which it was naturally produced). An “isolated” molecule may be in contact with other molecules (for example, as part of a reaction mixture). As used herein, “isolated” molecules also include recombinantly-produced proteins or nucleic acids which have an amino acid or nucleotide sequence which occurs naturally. “Isolated” nucleic acids include polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is at a chromosomal location different from that of natural cells. In some embodiments, “isolated” polypeptides are purified to at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% homogeneity as evidenced by SDS-PAGE of the polypeptides followed by Coomassie blue, silver, or other protein staining method.

As used herein, a nucleic acid molecule which is described as containing the “sequence” of a template or other nucleic acid may also be considered to contain the template or other nucleic acid itself (e.g. a molecule which is described as containing the sequence of a template may also be described as containing the template), unless the context clearly dictates otherwise.

As used herein, when a first polynucleotide is described as “annealed”, “annealing” or the like to a second polynucleotide, the entirety of the first polynucleotide or any portion thereof may anneal to the second polynucleotide, and vice versa.

As used herein, a reference to “treating” a given object to a condition or other object or the like refers to directly or indirectly exposing the given object to the recited condition or other object. Thus, while a “treating” step may involve a distinct related action (e.g. adding an enzyme to a vessel, shaking a vessel, etc.), not every “treating” step requires a distinct related action. For example, a reaction involving one or more reagents can be set up in a vessel, and once the reaction has been initiated, multiple events or steps may occur in the vessel without further human or mechanical intervention with the contents of the vessel. One or more of these multiple events or steps in the vessel may be described as “treating” an object in the vessel, even if no separate intervention with the contents of the vessel occurs after the initiation of the reaction.

Embodiments of methods and compositions provided herein may be described with reference to FIG. 1. A primary nucleic acid 101 may be provided (FIG. 1A). The primary nucleic acid 101 may contain the sequence of a nucleic acid template 102. The nucleic acid template 102 may be a double-stranded nucleic acid containing a first strand 103 and a second strand 104. In FIG. 1, the first strand 103 of the double-stranded nucleic acid template has an exemplary nucleotide sequence in the 5′ to 3′ direction of: AACGGTTGCTC (SEQ ID NO: 1). The first strand 103 also contains a 5′ terminal nucleotide (the 5′-most A) and a 3′ terminal nucleotide (the 3′-most C). The second strand 104 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: GAGCAACCGTT (SEQ ID NO: 2). The second strand 104 also contains a 5′ terminal nucleotide (the 5′-most G) and a 3′ terminal nucleotide (the 3′-most T).

A first primer 105 and a second primer 106 may also be provided (FIG. 1B). In FIG. 1, the first primer 105 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: ATGGGAGC (SEQ ID NO: 3). The first primer also contains a 5′ terminal nucleotide (the 5′-most A) and a 3′ terminal nucleotide (the 3′-most C). The second primer 106 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: CCATAACG (SEQ ID NO: 4). The second primer also contains a 5′ terminal nucleotide (the 5′-most C) and a 3′ terminal nucleotide (the 3′-most G). Although nucleic acid template 102 of FIG. 1 is 11 nucleotides in length and has an exemplary nucleotide sequence, these are exemplary, and not to be considered as limiting.

The first primer 105 may comprise two regions: i) a tail region and ii) a template-binding region (“temp” region). The tail region of the first primer 105 may contain three components: a) the 5′ terminal nucleotide of the primer, b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide, and c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides. In FIG. 1, the tail region of the first primer 105 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: ATGG, of which the 5′ terminal nucleotide of the primer is the A, the middle section is the middle TG, and the innermost nucleotide is the 3′-most G. The template-binding region of the first primer 105 may contain three components: a) the 3′ terminal nucleotide of the primer, b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide, and c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides. In FIG. 1, the template-binding region of the first primer 105 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: GAGC, of which the 3′ terminal nucleotide of the primer is the C, the middle section is the middle AG, and the innermost nucleotide is the 5′-most G.

The second primer 106 may comprise two regions: i) a tail region and ii) a template-binding region. The tail region of the first primer 106 may contain three components: a) the 5′ terminal nucleotide of the primer, b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide, and c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides. In FIG. 1, the tail region of the second primer 106 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: CCAT, of which the 5′ terminal nucleotide of the primer is the 5′-most C, the middle section is the middle CA, and the innermost nucleotide is the T. The template-binding region of the second primer 106 may contain three components: a) the 3′ terminal nucleotide of the primer, b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide, and c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides. In FIG. 1, the template-binding region of the second primer 106 has an exemplary nucleotide sequence in the 5′ to 3′ direction of: AACG, of which the 3′ terminal nucleotide of the primer is the G, the middle section is the middle AC, and the innermost nucleotide is the 5′-most A.

In some embodiments, the tail region of the second primer may contain a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, if the sequences of the primers are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer. For example, in FIG. 1, the tail region of the second primer 106 has a nucleotide sequence in the 5′ to 3′ direction of CCAT, and the tail region of the first primer 105 has a nucleotide sequence in the 5′ to 3′ direction of ATGG. These sequences are complementary when the 5′ terminal nucleotide of the second primer (C) is aligned with the innermost nucleotide of the tail region of the first primer (G) and the 5′ terminal nucleotide of the first primer (A) is aligned with the innermost nucleotide of the tail region of the second primer (T) 107 (FIG. 1B).

It should be noted that although the tail region of the second primer may contain a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer, typically, products formed by the annealing of the first primer to the second primer are not desirable or useful for methods or compositions provided herein. Accordingly, in some embodiments, steps may be taken to minimize the formation of first primer-second primer annealed products. Such steps may include, for example, not pre-incubating a first primer and a second primer under conditions where the primers may interact for an extended period of time before initiating a method provided herein.

The primary nucleic acid 101 may be treated with a first copy of the first primer 105 a and a polymerase 108 under conditions such that the template-binding region of the first copy of the first primer 105 a anneals to the first strand of the nucleic acid template 103 (FIG. 1C) and an extension product of the first copy of the first primer 111 is formed (FIG. 1D). The polymerase 108 may catalyze the formation of the extension product of the first copy of the first primer 111. The polymerase may have strand displacement activity. During the synthesis of the extension product of the first copy of the first primer 111, the polymerase may displace the second strand of the nucleic acid template 104 from the first strand of the nucleic acid template 103. Typically, the extension product is generated from the 3′ end of the first copy of the first primer 105 a. During the generation of the extension product of the first copy of the first primer 111, the first copy of the first primer 105 a may be covalently linked to the synthesized extension product, such that the first copy of the first primer becomes part of the molecule described herein as the “extension product of the first copy of the first primer”. The extension product of the first copy of the first primer 111 is complementary to the first strand of the nucleic acid template 103. In some embodiments, when the first copy of the first primer 105 a anneals to the first strand of the nucleic acid template 103, the template-binding region but not the tail region of the first copy of the first primer 105 a anneals to the first strand of the nucleic acid template.

In some embodiments, conditions such that the template-binding region of the first copy of the first primer 105 a anneals to the first strand of the nucleic acid template 103 and an extension product of the first copy of the first primer 111 is formed may include any condition sufficient to support polymerase-based nucleic acid synthesis. Example conditions for polymerase-based nucleic acid synthesis are known in the art and are provided, for example, in Molecular Cloning: A Laboratory Manual, M. R. Green and J. Sambrook, Cold Spring Harbor Laboratory Press (2012), which is herein incorporated by reference in its entirety. In embodiments, the template-binding region of the first primer or second primer may support template-dependent extension of the primer by a nucleic acid polymerase, which may extend the primer from the primer's 3′ end.

The extension product of the first copy of the first primer 111 may be treated with the second primer 106 and a polymerase 108 under conditions such that the template-binding region of the second primer 106 anneals to the extension product of the first copy of the first primer 111 (FIG. 1E) and an extension product of the second primer 112 is formed (FIG. 1F). The polymerase 108 may catalyze the formation of the extension product of the second primer 106. The polymerase may have strand displacement activity. The polymerase may be the same type of polymerase as is used to generate an extension product of the first copy of the first primer 111, or it may be different. During the synthesis of the extension product of the second primer 112, the polymerase may displace the first strand of the nucleic acid template 103 from the extension product of the first copy of the first primer 111. Typically, the extension product is generated from the 3′ end of the second primer 106. During the generation of the extension product of the second primer 112, the second primer 106 may be covalently linked to the synthesized extension product, such that the second primer becomes part of the molecule described herein as the “extension product of the second primer”. The extension product of the second primer 112 is complementary to the extension product of the first copy of the first primer 111. In some embodiments, when the second primer 106 anneals to the extension product of the first copy of the first primer 111, the template-binding region but not the tail region of the second primer 106 anneals to the extension product of the first copy of the first primer 111. The extension product of the second primer 112 contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region. The final nucleotide of the 3′ terminal region (T) is the 3′ terminal nucleotide of the extension product of the second primer (T), and the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the second primer read in the 5′ to 3′ direction (CCAT).

In some embodiments, conditions such that the template-binding region of the second primer 106 anneals to the extension product of the first copy of the first primer 111 and an extension product of the second primer 112 is formed may include any condition sufficient to support polymerase-based nucleic acid synthesis, including any condition discussed elsewhere herein. The conditions may be the same as used to generate an extension product of the first copy of the first primer 111, or they may be different.

The extension product of the second primer 112 may be treated with a second copy of the first primer 105 b and a polymerase 108 under conditions such that the template-binding region of the second copy of the first primer 105 b anneals to the extension product of the second primer 112 (FIG. 1G) and an extension product of the second copy of the first primer 113 is formed (FIG. 1H). The polymerase 108 may catalyze the formation of the extension product of the second copy of the first primer 113. The polymerase may have strand displacement activity. The polymerase may be the same type of polymerase as is used to generate an extension product of the first copy of the first primer 111 or an extension product of the second primer 112, or it may be different. During the synthesis of the extension product of the second copy of the first primer 113, the polymerase may displace the extension product of the first copy of the first primer 111 from the extension product of the second primer 112. Typically, the extension product is generated from the 3′ end of the second copy of the first primer 105 b. During the generation of the extension product of the second copy of the first primer 113, the second copy of the first primer 105 b may be covalently linked to the synthesized extension product, such that the second copy of the first primer becomes part of the molecule described herein as the “extension product of the second copy of the first primer”. The extension product of the second copy of the first primer 113 is complementary to the extension product of the second primer 112. In some embodiments, when the second copy of the first primer 105 b anneals to the extension product of the second primer 112, both the template-binding region and the tail region of the second copy of the first primer 105 b anneals to the extension product of the second primer 112. The extension product of the second copy of the first primer 113 contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region. The final nucleotide of the 3′ terminal region (G) is the 3′ terminal nucleotide of the extension product of the second primer (G), and the 3′ terminal region contains the same nucleotide sequence as the nucleotide sequence of the tail region of the first primer read in the 5′ to 3′ direction (ATGG).

In some embodiments, conditions such that the template-binding region of the second copy of the first primer 105 b anneals to the extension product of the second primer 112 and an extension product of the second copy of the first primer 113 is formed may include any condition sufficient to support polymerase-based nucleic acid synthesis, including any condition discussed elsewhere herein. The conditions may be the same as used to generate an extension product of the first copy of the first primer 111 or an extension product of the second primer 112, or they may be different.

Generation of the extension product of the second copy of the first primer 113 may result in the generation of a molecule comprising the extension product of the second copy of the first primer 113 and the extension product of the second primer 112, which may be referred to herein as the “secondary nucleic acid” 115. Within the secondary nucleic acid 115, the extension product of the second copy of the first primer 113 may be annealed to the extension product of the second primer 112. In addition, the secondary nucleic acid 115 contains the sequence of the nucleic acid template 102. The secondary nucleic acid 115 has a greater nucleotide length than the nucleic acid template 102 alone, as in addition to the sequence of the nucleic acid template 102, it may include the sequence of the tail regions of the first primer and the second primer, and complementary sequences thereof. Specifically, a secondary nucleic acid 115 may have a first end region 116 and a second end region 117. The first end region 116 may comprise the 3′ terminal region of the extension product of the second primer 112, and the complement thereof. The second end region 117 may comprise the 3′ terminal region of the extension product of the second copy of the first primer 113, and the complement thereof.

A first copy 115 a and a second copy 115 b of the secondary nucleic acid 115 may be formed or provided (FIG. 1I). The first copy 115 a and second copy 115 b of the secondary nucleic acid 115 may be generated by any process whereby a nucleic acid having the general structure of the secondary nucleic acid 115 may be generated, including any process discussed elsewhere herein. For example, the full process as described above for generating a secondary nucleic acid 115 from a primary nucleic acid 101 may be repeated two times, in order to generate the first copy 115 a and the second copy 115 b of the secondary nucleic acid. In another example, the first copy 115 a and the second copy 115 b of the secondary nucleic acid may be generated from a single copy of an extension product of a first copy of first primer 111. In this example, two copies 112 a, 112 b of an extension product of the second primer may be generated from the single copy of the extension product of a first copy of a first primer 111, which may occur if a first copy of the extension product of the second primer 112 a is displaced from the single copy of the extension product of the first copy of the first primer 111, thereby permitting the generation of a second copy of the extension product of the second primer 112 b from the single copy of the extension product of a first copy of a first primer 111. Then, for example, an extension product of the second copy of the first primer 113 a, 113 b may be generated from each copy of the extension product of the second primer 112 a, 112 b, respectively. Additional procedures for the generation of a first copy 115 a and a second copy 115 b of the secondary nucleic acid may also be performed and used with methods provided herein.

The first copy 115 a and second copy 115 b of the secondary nucleic acid may be treated under conditions such that the 3′ terminal region of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid 113 a anneals to the 3′ terminal region of the extension product of the second primer of the second copy of the secondary nucleic acid 112 b, to produce a cross-over structure 118 comprising these strands (FIG. 1J) (for simplicity, strands 113 b and 112 a are not shown). The cross-over structure 118 may be further treated with a polymerase under conditions such that extension products of both component strands 112 b, 113 a are formed, the extension products which may be referred to herein as a “first concatemer strand” 121 and a “second concatemer strand” 122, respectively. The first concatemer strand 121 and the second concatemer strand 122 may be annealed together, and may be collectively referred to as a concatemer 120 (FIG. 1K). The concatemer 120 may contain two or more copies of the nucleic acid template 102. Within the concatemer 120, some or all of the two or more copies of the nucleic acid template 102 may be separated from each other by the sequences of the tail regions of the first primer 105 and second primer 106, where the sequences of the tail regions are annealed to each other. For example, in the concatemer 120 of FIG. 1K, the first copy of the first strand of the double-stranded nucleic acid template is separated from the second copy of the first strand of the double-stranded nucleic acid template by the sequence, in 5′-3′ order: CCAT. CCAT is also the sequence, in 5′-3′ order, of the tail region of the second primer 106. Similarly, in the concatemer 120 of FIG. 1K, the first copy of the second strand of the double-stranded nucleic acid template is separated from the second copy of the second strand of the double-stranded nucleic acid template by the sequence, in 5′-3′ order: ATGG. ATGG is also the sequence, in 5′-3′ order, of the tail region of the first primer 105. Within the concatemer, the CCAT of the first concatemer strand is annealed to the ATGG of the second concatemer strand.

In some embodiments, conditions such that a cross-over structure 118 or extension products of the strands of a cross-over structure are formed may include any condition sufficient to support polymerase-based nucleic acid synthesis, including any condition discussed elsewhere herein. The conditions may be the same as used to generate an extension product of the first copy of the first primer 111, an extension product of the second primer 112, or an extension product of the second copy of the first primer 111, or they may be different.

In some embodiments, concatemers provided herein may further increase in length according to the process generally outlined in FIG. 1I-1K. For example, two of the concatemer molecules 120 as shown in FIG. 1K may be treated such that they form a cross-over structure similar to that shown in FIG. 1J (except with longer strands), followed by generation of a larger concatemer molecule containing four copies of the nucleic acid template 102. In another example, a secondary nucleic acid 115 and a concatemer 120 may form a cross-over structure, followed by generation of a larger concatemer molecule containing three copies of the nucleic acid template 102. In some embodiments, in methods provided herein, multiple different concatemers of multiple different lengths may be simultaneously generated.

In embodiments, methods and compositions provided herein may be described with reference to FIG. 8. A polynucleotide template 800 (FIG. 8A) may be a target for amplification. The polynucleotide template 800 is a single nucleic acid strand. The polynucleotide template strand may exist as a free, single-stranded molecule, or it may exist as part of a double-stranded molecule, in which the polynucleotide template strand is annealed to a complementary strand thereof. The polynucleotide template 800 may be DNA or RNA.

The polynucleotide template 800 may contain at least a first portion 801 (also indicated with an “(i)”) and a second portion 802 (also indicated with an “(ii)”). The first portion 801 and second portion 802 may each contain any number of nucleotides, such as less than 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides each or at least 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides each. Typically, the first portion 801 and second portion 802 may each contain between 10 and 30 nucleotides, although the first portion 801 and second portion 802 may be other lengths as described elsewhere herein, as well. The first portion 801 and second portion 802 may contain the same or a different number of nucleotides. In some embodiments, the polynucleotide template 800 may contain other portions in addition to the first portion 801 and second portion 802. In other embodiments, the first portion 801 and second portion 802 may constitute the entirety of the polynucleotide template 800. The polynucleotide template 800 may have the length of a nucleic acid template described elsewhere herein. The polynucleotide template 800 may be a portion of a primary nucleic acid. The polynucleotide template may be present in a sample obtained from a subject.

The polynucleotide template 800 may be incubated in a reaction mixture containing a first primer 810 and a second primer 820 (FIG. 8B). Typically, the reaction mixture contains multiple copies of each primer, such as, for example, at least 4, 5, 6, 10, 15, 20, 50, 100, 1000, 10,000, 100,000, or 1 million copies or more of each of the primers. The first primer 810 may have a first region 811 (also indicated with an “(i)”) and a second region 812 (also indicated with an “(ii)”). The first region of the first primer 811 may have a 5′ end, and the second region of the first primer 812 may have a 3′ end. The first region of the first primer may have a nucleotide sequence which is different from the nucleotide sequence of the second region of the first primer. The second primer 820 may have a first region 821 (also indicated with a “(i)”) and a second region 822 (also indicated with a “(ii)”). The first region of the second primer 821 may have a 5′ end, and the second region of the second primer 822 may have a 3′ end. The first region of the second primer may have a nucleotide sequence which is different from the nucleotide sequence of the second region of the second primer.

The second region of the first primer 812 may have a nucleotide sequence which is complementary to the nucleotide sequence of the first portion of the polynucleotide template 801. In FIG. 8, nucleotide sequence of the first portion of the polynucleotide template 801 is represented by the letter A, and the nucleotide sequence of the second region of the first primer 812 is represented by the letter A′, where A and A′ are complementary to each other. The second region of the second primer 822 may have a nucleotide sequence which is complementary to a partner nucleotide sequence 830, wherein the partner nucleotide sequence 830 is complementary to the nucleotide sequence of the second portion of the polynucleotide template 802. In FIG. 8, the nucleotide sequence of the second portion of the polynucleotide template 802 is represented by the letter B, the nucleotide sequence of the partner nucleotide sequence 830 is represented by the letter B′, and the nucleotide sequence of the second region of the second primer 822 is represented by the letter B, where B and B′ are complementary to each other. In other words, as indicated by FIG. 8, the nucleotide sequence of the second portion of the polynucleotide template 802 (B) may be the same as the nucleotide sequence of the second region of the second primer 822 (B). With compositions and methods provided herein, a partner nucleotide sequence 830 may be generated, for example, as part of an extension product from the 3′ end of the first primer. In embodiments, the first region of the first primer 811 may be complementary to the first region of the second primer 821. In FIG. 8, the nucleotide sequence of the first region of the first primer is represented by the letter C, and the nucleotide sequence of the first region of the second primer is represented by the letter C′, where C and C′ are complementary to each other.

In embodiments, the first primer and second primer of FIG. 8 may have any of the characteristics of the first primer and second primer described elsewhere herein (e.g. as in FIG. 1), respectively. Also, in embodiments, the first region of the first primer of FIG. 8 may have any of the characteristics of the tail region of a first primer described elsewhere herein. In embodiments, the second region of the first primer of FIG. 8 may have any of the characteristics of the template binding region of a first primer described elsewhere herein. In embodiments, the first region of the second primer of FIG. 8 may have any of the characteristics of the tail region of a second primer described elsewhere herein. In embodiments, the second region of the second primer of FIG. 8 may have any of the characteristics of the template binding region of a second primer described elsewhere herein.

In embodiments, a reaction mixture containing a polynucleotide template 800 and at least two copies of a first primer 810 and at least two copies of a second primer 820 may contain one or more other components which may support polymerase-based nucleic acid synthesis as described elsewhere herein (e.g. polymerases, nucleotides, buffers, water, etc.). Similarly, a reaction mixture containing a polynucleotide template 800 and at least two copies of a first primer 810 and at least two copies of a second primer 820 may be incubated at any of the reaction conditions described elsewhere herein for supporting the amplification of a nucleic acid template or the generation of concatemers.

In embodiments, upon incubation of a polynucleotide template 800 and at least two copies of a first primer 810 and at least two copies of a second primer 820 in a reaction mixture as described herein, one or more concatemer strands may be formed (FIG. 8C). In embodiments, a formed concatemer strand 840 may have a 5′ end and a 3′ end, and may have a sequence containing the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein C′ is the nucleotide sequence of the first region of the second primer, T is the sequence of the polynucleotide template or an analogous sequence thereof, and X is any number and sequence of nucleotides. Thus, as indicated by the general structure of the concatemer strand, a concatemer strand may contain at least two copies of the sequence of the polynucleotide template or an analogous sequence thereof, where the copies of the sequence of the polynucleotide template are separated by at least the sequence of the first region of the second primer. In embodiments, T may be an analogous sequence of the polynucleotide template. As used herein, an “analogous sequence” of a polynucleotide template refers to a sequence which contains a similar sequence as the polynucleotide template, but which contains one or more analogous nucleotides to a nucleotide in the polynucleotide template. Thus, for example, if the polynucleotide template contains an RNA sequence, an analogous sequence thereof may be a DNA version of the same sequence (i.e. the uracils in the RNA sequence are thymines in the analogous DNA sequence), or vice-versa. Continuing with the example, if the polynucleotide template contains an RNA sequence of 5′ UACCUG 3′, an analogous sequence is the DNA sequence of 5′ TACCTG 3′.

In embodiments, in a sequence containing the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, X is zero nucleotides (i.e. the T and C sequence on either side of the X are directly linked). In other embodiments, X is less than 10,000, 5000, 4000, 3000, 2000, 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides. The nucleotides (if any) in X may have any sequence. In embodiments, X contains a sequence having the general structure in the 5′ to 3′ direction of [(C′-T)_(N)] wherein C′ is the nucleotide sequence of the first region of the second primer, T is the sequence of the polynucleotide template or analogous sequence thereof, and N is any integer less than 1000, 500, 200, 100, 50, or 10, such as an integer between 1 and 500, 1 and 200, 1 and 100, 1 and 50, or 1 and 10. For example, if “N” is 3 when X contains a sequence having the general structure in the 5′ to 3′ direction of [(C′-T)_(N)], there are 3 sequential repeats of (C′-T) in the X position of the structure C′-T-C′-T-X-C′, such that the corresponding structure could be written as: C′-T-C′-T-C′-T-C′-T-C′-T-C′, where the underlined C's and Ts are the C′-T repeats of the formula [(C′-T)₃]. Thus, for example, according to compositions and methods provided herein, concatemer strands may be formed which contain tens or hundreds of copies of the polynucleotide template. In embodiments, a concatemer strand as provided in FIG. 8 may have any of the characteristics of a concatemer strand described elsewhere herein. Also, in some embodiments, during the generation of a concatemer strand, at one or more positions of a C′ or Tin a sequence having the general structure C′-T-C′-T-X-C′, a small number of nucleotides (e.g. 15 or less, 10 or less, 5 or less, or 3 or less) may be inserted between a C′ and T. Also, in some embodiments, during the generation of a concatemer strand, at one or more positions of a C′ or Tin a sequence having the general structure C′-T-C′-T-X-C′ or a small number of nucleotides (e.g. 15 or less, 10 or less, 5 or less, or 3 or less) may be deleted from a C′ or T sequence. Thus, concatemer strands generated according to methods provided herein may include strands which have a general structure as provided herein, but which may have a small number of nucleotides added or removed at one or more junction points between different sequence components of the concatemer strand (i.e. junctions between template sequence and primer sequence/T and C′). Also, in embodiments, a concatemer strand as provided herein may contain additional nucleotides (e.g. up to 3, 5, 10, 20, 50, 100, 500, 1000 or more additional nucleotides) at the 5′ or 3′ end of a molecule containing a concatemer having a sequence with the general structure C′-T-C′-T-X-C′. In embodiments, a C′ of the general structure C′-T-C′-T-X-C′ may have any of the characteristics (e.g. length, sequence) of the tail region of a second primer described elsewhere herein. In embodiments, a T of the general structure C′-T-C′-T-X-C′ may have any of the characteristics (e.g. length, sequence) of a polynucleotide template strand described elsewhere herein.

In embodiments, a concatemer strand 840 generated according to method provided herein may be part of a single-stranded molecule. In other embodiments, a concatemer strand 840 generated according to method provided herein may be part of a double-stranded concatemer, in which the concatemer strand 840 is annealed to a strand which is complementary to the concatemer strand 850. While FIG. 8C depicts concatemer strands having a sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′ (strand 840) or C-T′-C-T′-X′-C (strand 850), wherein X is 0 nucleotides, a concatemer strand generated according to the schematic of FIG. 8 may have any of the characteristics of a concatemer strand described above or elsewhere herein. Also, as relating to FIG. 8C, sequences A and B are part of the template strand, and thus are not separately notated in the general structure C′-T-C′-T-X-C′ or C-T′-C-T′-X′-C.

To further indicate the relationship of elements in embodiments of FIG. 8, blocks 860, 862, 864, 866, and 868 are provided in FIG. 8. Block 860 (with a backslash-like fill) indicates the relative position of the total polynucleotide template sequence (i.e. element 800), or its complementary sequence. Block 862 (with vertical line fill) indicates the relative position of the sequence of first portion of the polynucleotide template (i.e. the relative position of the sequence of element 801), or its complementary sequence. Thus, block 862 also represents the sequence of the second region of the first primer (i.e. element 812), which is complementary to element 801. Block 864 (with vertical and horizontal line fill) indicates the relative position of the sequence of the second portion of the polynucleotide template (i.e. the relative position of the sequence of element 802), or its complementary sequence. Thus, block 864 also represents the sequence of the partner nucleotide sequence (i.e. element 830), which is complementary to element 802. Block 864 also indicates the relative position of the sequence of the second region of the second primer 822, which may be the same sequence as the sequence of the second portion of the polynucleotide template (element 802), or a similar sequence. In other words, in embodiments the nucleotide sequence of 822 may be the same as or similar to nucleotide sequence of 802. As depicted, for example, in FIG. 8A, in embodiments, there are nucleotides in the polynucleotide template which are not part of the first portion or the second portion of the polynucleotide template. For instance, in one example, the first portion of the polynucleotide template may contain 15 nucleotides, the second portion of the polynucleotide template may contain 15 nucleotides, and between the first portion and second portion of the polynucleotide template are 25 nucleotides. Typically, the first portion of the polynucleotide template and the second portion of the polynucleotide template are each between 10 and 30 nucleotides in length, although other numbers are also contemplated. For example, each of the first portion and second portion of the polynucleotide template may be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 100 nucleotides, no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 100 nucleotides, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides and no more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 100 nucleotides (with the upper limit being above the lower limit). In embodiments, in the polynucleotide template between the first portion and second portion, there may be, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 500 nucleotides, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 500 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, or 200 nucleotides and no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 500 nucleotides (with the upper limit being above the lower limit). Block 866 (with wavy line fill) indicates the relative position of the sequence of the first region of the first primer (i.e. the relative position of the sequence of element 811/region “C”), or its complementary sequence. Thus, block 866 also represents the sequence of the first region of the second primer (element 821/region “C′”), which is complementary to element 811. Block 868 (with diamond fill) indicates the relative position of the sequence of the concatemer strand (element 840), or its complementary sequence. Thus, block 868 also represents the sequence of a strand which is complementary to the concatemer strand (element 850). As depicted in FIG. 8C, within the concatemer strand or its complement (868), two copies of the polynucleotide template sequence or its complement (860) are present, separated by the nucleotide sequence of the first region of the first primer or its complement (866). In addition, within the concatemer strand or its complement (868), the nucleotide sequence of the first region of the first primer or its complement (866) flank the polynucleotide template sequence or its complement (860) on both ends. Also, within the concatemer strand or its complement (868), there are two copies of the sequence of the second region of the first primer or its complement (862) and two copies of the sequence of the second region of the second primer or its complement (864), which are both present in two copies as part of the two copies of the polynucleotide template sequence or its complement. Blocks 860, 862, 864, 866, and 868 are not to be interpreted to as to dictate any particular required size relationship between the elements of FIG. 8; rather blocks 860, 862, 864, 866, and 868 are simply to help indicate the general position of the elements of FIG. 8 relative to each other in certain embodiments provided herein.

Concatemers generated according to methods and compositions provided herein may be of any length of nucleotides. In some embodiments, concatemer molecules generated herein may be at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000 nucleotides in length. In some embodiments, concatemer molecules generated herein may be no more than 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000 nucleotides in length. In some embodiments, concatemer molecules generated herein may have a length selected from a range having a minimum value of 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, or 20,000 nucleotides in length, and a maximum value of 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000 nucleotides in length. In some embodiments, at least some concatemers generated according to a method or composition provided herein have characteristics described above. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of concatemers generated according to a method or composition provided herein have characteristics described above.

Concatemers generated according to methods and compositions provided herein may contain any number of copies of a nucleic acid template or particular nucleic acid. In some embodiments, concatemer molecules generated herein may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies of a nucleic acid template or particular nucleic acid. In some embodiments, concatemer molecules generated herein may contain no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies of a nucleic acid template or particular nucleic acid. In some embodiments, concatemer molecules generated herein may have a number of copies of a nucleic acid template or particular nucleic acid selected from a range having a minimum value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 copies, and a maximum value of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies. In some embodiments, at least some concatemers generated according to a method or composition provided herein have characteristics described above. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of concatemers generated according to a method or composition provided herein have characteristics described above.

Progress of a method provided herein may be monitored in multiple different ways. In embodiments, a reaction may be assayed for a nucleic acid amplification product (e.g. for the amount of the product or the rate of its generation). In other embodiments, a reaction may be assayed for the activity of a polymerase along a nucleic acid template (e.g. for movement of a polymerase along a template strand). Thus, in some embodiments, events of a method provided herein may observed due to the accumulation of product from a method (which may be during or after completion of steps of the method), or due to detectable events occurring during the steps of a method.

The presence of amplified nucleic acids can be assayed, for example, by detection of reaction products (amplified nucleic acids or reaction by-products) or by detection of probes associated with the reaction progress.

In some embodiments, reaction products may be identified by staining the products with a dye. In some embodiments, a dye may have greater fluorescence when bound to a nucleic acid than when not bound to a nucleic acid. In embodiments, a dye may intercalate with a double-stranded nucleic acid or it may bind to an external region of a nucleic acid. Nucleic acid dyes that may be used with methods and compositions provided herein include, for example, cyanine dyes, PicoGreen®, OliGreen®, RiboGreen®, SYBR® dyes, SYBR® Gold, SYBR® Green I, SYBR® Green II, ethidium bromide, dihydroethidium, BlueView™, TOTO® dyes, TO-PRO® dyes, POPO® dyes, YOYO® dyes, BOBO® dyes, JOJO® dyes, LOLO® dyes, SYTOX® dyes, SYTO® dyes, propidium iodide, hexidium iodide, methylene blue, DAPI, acridine orange, quinacrine, acridine dimers, 9-amino-6-chloro-2-methoxyacridine, bisbenzimide dyes, Hoechst dyes, 7-aminoactinomycin D, actinomycin D, hydroxystilbamidine, pyronin Y, Diamond™ dye, GelRed™, GelGreen™ and LDS 751.

In some embodiments, reaction products may be identified by analysis of turbidity of amplification reactions. For example, in embodiments, increased turbidity may be correlated with formation of reaction products and reaction by-products (e.g. pyrophosphate complexed with magnesium).

In some embodiments, reaction products may be identified by separating a reaction performed according to a method herein by gel electrophoresis, followed by staining of the gel with a dye for nucleic acids. The dye may be any nucleic acid dye disclosed herein or otherwise known in the art.

In some embodiments, any method or composition known in the art for the detection of nucleic acids or for the generation of nucleic acids may be used with methods and compositions provided herein.

In some embodiments, a nucleic acid probe which contains a nucleotide sequence complementary to a portion of a nucleic acid template strand (or a strand having a similar or identical sequence) and which contains one or both of a fluorescent reporter (fluorophore) and a quencher are included in a reaction provided herein.

In an example, a nucleic acid probe may contain a fluorescent reporter at its 5′ or 3′ terminus, and a quencher at the other terminus. The probe may further have a nucleotide sequence containing, in order, at least a first, second, and third region, where the first and third regions are complementary to each other, and where at least a portion of the second region is complementary to a portion of a strand of the nucleic acid template (the probe “detection sequence”). In some embodiments, the length of the second region may be greater than the length of the first or third regions. In some embodiments, the length of the second region may be between 10 and 40 nucleotides, and the length of first and third regions may be between 4 and 10 nucleotides. The probe may have at least two different conformations: (A) a conformation where the probe is not annealed to its detection sequence and where the first and third regions are annealed to each other; this conformation may be a “stem-loop” structure, where the first and third regions form the stem and the second region forms the loop, and (B) a conformation where the probe is annealed to its detection sequence; in this conformation, the second region or a portion thereof is annealed to its detection sequence and the first and third regions are not annealed to each other. In conformation (A) of the probe, the fluorescent reporter and quencher (which are located at opposite termini of the probe/at the outer ends of the first and third regions) may be in close proximity to each other (both being at the end of the stem structure formed by the annealing of the first and third regions), such that the fluorescent reporter is quenched. In conformation (B) of the probe, the fluorescent reporter and quencher may not be in close proximity to each other, such that the fluorescent reporter is not quenched. The probe may be used to monitor accumulation of a selected reaction product, for example, under reaction conditions where the probe may either form a stem-loop structure or anneal to its detection sequence. In some embodiments, if the detection sequence is present, the probe may anneal to the detection sequence, and the probe may fluoresce in response to light of a wavelength of the fluorophore's excitation spectrum. In contrast, if the detection sequence is not present, the probe may form a stem-loop structure, and not fluoresce in response to light of a wavelength of the fluorophore's excitation spectrum.

In another example, a nucleic acid probe may contain a fluorescent reporter at its 5′ or 3′ terminus, and it may be annealed to a nucleic acid primer containing a quencher. The nucleic acid primer containing a quencher may contain the quencher at a position in the primer such that when the nucleic acid probe is annealed to the primer, the fluorescent reporter is quenched. The probe may be used to monitor accumulation of a selected reaction product, for example, under reaction conditions where the probe may either anneal to the primer or anneal to its detection sequence in the reaction product. In some embodiments, if the detection sequence is present, the probe may anneal to the detection sequence, and the probe may fluoresce in response to light of a wavelength of the fluorophore's excitation spectrum (thus indicating the presence of the reaction product). In contrast, if the detection sequence is not present, the probe may remain paired with the primer, and not fluoresce in response to light of a wavelength of the fluorophore's excitation spectrum.

In probes containing a fluorescent reporter and quencher pair, the fluorescent reporter and quencher may be selected so that the quencher can effectively quench the reporter. In some embodiments, a fluorescent reporter is paired with a quencher where the emission maximum of the fluorescent reporter is similar to the absorption maximum of the quencher. Fluorophores that may be used as the fluorescent reporter include, for example, CAL Fluor Gold, CAL Fluor Orange, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 670 (Biosearch Technologies), VIC, NED (Life Technologies), Cy3, Cy5, Cy5.5 (GE Healthcare Life Sciences), Oyster 556, Oyster 645 (Integrated DNA Technologies), LC red 610, LC red 610, LC red 640, LC red 670, LC red 705 (Roche Applies Science), Texas red, FAM, TET, HEX, JOE, TMR, and ROX. Quenchers that may be used include, for example, DDQ-I, DDQ-II (Eurogentec), Eclipse (Epoch Biosciences), Iowa Black FQ, Iowa Black RQ (Integrated DNA Technologies), BHQ-1, BHQ-2, BHQ-3 (Biosearch Technologies), QSY-7, QSY-21 (Molecular Probes), and Dabcyl.

In some embodiments, a probe used with a composition or method provided herein may be a peptide nucleic acid (PNA) probe. Peptide nucleic acids are synthetic DNA analogs in which, for example, the phosophodiester backbone is replaced by repetitive units of N-(2-aminoethyl) glycine and to which the purine and pyrimidine bases are attached via a methyl carbonyl linker. Optionally, a PNA probe may be provided in a reaction provided herein which further contains a dye which binds to PNA-DNA duplexes, and which changes color upon binding to PNA-DNA duplexes. An example of such a dye is 3,3′-diethylthiadicarbocyanine iodide [DiSc₂(5)]. DiSc₂(5) changes color from blue to purple upon binding to PNA-DNA duplexes, and this color change may be observed, for example in a spectrophotometer, by image analysis, or by an unaided human eye. The use of PNA probes and DiSc₂(5) for detection of PNA-DNA duplexes is described, for example in Wilhelmsson et. al, Nucleic Acids Research, 2002, Vol. 30, No. 2 e3, which is hereby incorporated by references for all purposes. In embodiments using a PNA probe, typically, the PNA probe will be designed to be complementary to a sequence that is to be amplified in the relevant amplification reaction. Thus, if the sequence is successfully amplified, the PNA probe will anneal to DNA strands containing the amplified sequence, and this annealing can be detected (e.g. by observing the color change in DiSc₂(5) when it binds to PNA-DNA duplexes). Use of a PNA probe with embodiments provided herein thus may aid in accurately determining reactions in which a sequence is successfully amplified, and in differentiating these reactions from reactions which have non-specific amplification of nucleic acids. To use a PNA probe with embodiments provided herein, optionally, the PNA probe may be included in a reaction mixture provided herein at the start of the reaction. In other examples, the PNA probe may be added to the reaction mixture at a time after the initiation of the reaction or after the completion of the reaction. A dye may be added to a reaction mixture at the same time a PNA probe is added, or at a different time. For example, in embodiments, a dye may be added to the reaction mixture at the start of the reaction, and the PNA may be added at point after the initiation of the reaction. In embodiments, a kit provided herein may further contain a PNA probe which is complementary to a nucleotide sequence to be amplified by reagents of the kit, and dye which binds to PNA-DNA duplexes.

In some embodiments, a reaction performed according to a method provided herein may be monitored in an apparatus containing a light source and an optical sensor. In some situations, the reaction may be positioned in the path of light from the light source, and light absorbed by the sample (e.g. in the case of a turbid reaction), scattered by the sample (e.g. in the case of a turbid reaction), or emitted by the sample (e.g. in the case of a reaction containing a fluorescent molecule) may be measured. In some embodiments, a method provided herein may be performed or monitored in a device or module therein as disclosed in U.S. patent application Ser. No. 13/769,779, filed Feb. 18, 2013, which is herein incorporated by reference in its entirety.

Using methods provided herein, specific amplification products of a nucleic acid template of interest may be identified within, for example, 30 seconds, 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes of initiation of an amplification reaction. In other examples, using methods provided herein, amplification reactions which are positive for a nucleic acid template of interest may be identified when as few as 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 copies of the template are generated. In other examples, using methods provided herein, the presence of a nucleic acid template of interest in a sample containing as few as 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000, 5000, or 10,000 copies of the template of interest at the start of the method may be identified.

In embodiments, methods provided herein may be used to assay a sample for a target nucleic acid of interest. In certain embodiments, the presence or quantity of a target nucleic acid of interest in a sample may be determined by a method involving determining an inflection time for nucleic acid amplification in a reaction. An inflection time/inflection point is a time or a point where an amplification reaction is determined as being positive for a nucleic acid template. An inflection time/point may be identified by one or more indicators, such as for example, the time post-initiation of a reaction when a selected quantity of nucleic acid has been generated in the reaction, the time when the rate of amplification in a reaction changes from a baseline phase to an exponential phase, or the time when the rate of amplification in a reaction changes from an exponential phase to a plateau phase, etc. In embodiments, an inflection time/point may be identified based on a change in fluorescence or absorbance of a reaction, or upon the fluorescence or absorbance of a reaction reaching a selected value. In certain embodiments, the presence or quantity of a target nucleic acid of interest in a sample may be determined by a method involving comparison of an inflection time for nucleic acid amplification of a reaction of which has an unknown amount of target nucleic acid of interest versus one or both of: i) a reaction which is known to lack the target nucleic acid of interest (i.e. a negative control) or ii) a reaction which is known to contain the target nucleic acid of interest (i.e. a positive control). In embodiments, both a reaction which contains the target nucleic acid of interest and a reaction which does not contain the target nucleic acid may be measured for a selected inflection time. In embodiments, the presence of a target nucleic acid of interest in a sample may be determined based on a method which involves evaluation of the difference in time between inflection of a reaction containing a sample which may or may not contain a target nucleic acid of interest, and a time of inflection of one or more reactions with known target nucleic acid of interest status (e.g. which are known to contain or not contain the target nucleic acid of interest). For example, a sample may be identified as containing a target nucleic acid of interest if the inflection time of the reaction according to a method provided herein is at least 3, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes earlier than a corresponding reaction which is known to not contain the target nucleic acid of interest. In another example, a sample may be identified as containing a target nucleic acid of interest if the inflection time of the reaction according to a method provided herein is no more than 3, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes later than a corresponding reaction which is known to contain the target nucleic acid of interest.

Methods provided herein may be performed for any length of time. Typically, the method will be performed for a length of time sufficient to monitor, for example, the rate of nucleic acid replication, the occurrence of polymerase activity, or the accumulation of amplification product. In some embodiments, a method provided herein may be performed for a total of less than 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours, by which time the rate of nucleic acid replication, the occurrence of polymerase activity, or the accumulation of amplification product is measured.

Methods provided herein may be terminated in various ways. In one embodiment, steps of a method may end upon the reduction in concentration or complete consumption of one or more reagents involved in one or more steps of the method (e.g. dNTPs). In another embodiment, steps of a method may end upon inactivation of one or more enzymes involved in one or more steps of the method (e.g. polymerases). Enzymes may be inactivated by various ways. For example, enzymes may gradually lose enzymatic activity over time due to random events that affect the structure of the enzyme, or enzymes may be exposed to a condition to accelerate the inactivation of the enzyme activity (e.g. high heat, extreme pH, etc.).

In some embodiments, a primary nucleic acid may be single stranded or double-stranded. A single stranded primary nucleic acid may also be referred to herein as a “primary polynucleotide”. A primary nucleic acid may be linear or circular. A primary nucleic acid may comprise a nucleic acid template. In some embodiments, the entirety of a primary nucleic acid may be a nucleic acid template. In other embodiments, a primary nucleic acid may contain one or more nucleotides which are not part of a nucleic acid template (e.g. the primary nucleic acid may be of a greater length than a nucleic acid template contained within the primary nucleic acid). In some embodiments, a primary nucleic acid may contain two or more copies of a nucleic acid template. A primary nucleic acid may contain DNA, RNA, or a mixture thereof. A double-stranded linear primary nucleic acid may have blunt ends or sticky ends (“sticky ends” refer to ends having an overhanging strand having one or more unpaired nucleotides).

A primary nucleic acid may be of any length of nucleotides. For example, a primary nucleic acid may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In another example, a primary nucleic acid may be between 2 and 100,000, between 5 and 100,000, between 10 and 100,000, between 15 and 100,000, between 20 and 100,000, between 25 and 100,000, between 30 and 100,000, between 50 and 100,000, between 70 and 100,000, between 100 and 100,000, between 200 and 100,000, between 2 and 10,000, between 5 and 10,000, between 10 and 10,000, between 15 and 10,000, between 20 and 10,000, between 25 and 10,000, between 30 and 10,000, between 50 and 10,000, between 70 and 10,000, between 100 and 10,000, between 200 and 10,000, between 2 and 5,000, between 5 and 5,000, between 10 and 5,000, between 15 and 5,000, between 20 and 5,000, between 25 and 5,000, between 30 and 5,000, between 50 and 5,000, between 70 and 5,000, between 100 and 5,000, between 200 and 5,000, between 2 and 3,000, between 5 and 3,000, between 10 and 3,000, between 15 and 3,000, between 20 and 3,000, between 25 and 3,000, between 30 and 3,000, between 50 and 3,000, between 70 and 3,000, between 100 and 3,000, between 200 and 3,000, between 2 and 1,000, between 5 and 1,000, between 10 and 1,000, between 15 and 1,000, between 20 and 1,000, between 25 and 1,000, between 30 and 1,000, between 50 and 1,000, between 70 and 1,000, between 100 and 1,000, between 200 and 1,000, between 2 and 500, between 5 and 500, between 10 and 500, between 15 and 500, between 20 and 500, between 25 and 500, between 30 and 500, between 50 and 500, between 70 and 500, between 100 and 500, or between 200 and 500 nucleotide bases in length.

In some embodiments, a nucleic acid template may be single stranded or double-stranded. A single strand of a nucleic acid template may be referred to herein as a “polynucleotide template”. A “polynucleotide template” as referred to herein is not precluded from binding to a complementary sequence thereof. In other words, a “polynucleotide template” may be, for example, the entirety of a single-stranded nucleic acid template, or it may be one strand of a double-stranded nucleic acid template. A nucleic acid template may be contained in a primary nucleic acid molecule. In some embodiments, a nucleic acid template may constitute the entirety of a primary nucleic acid molecule. In other embodiments, a nucleic acid template may be contained in a primary nucleic acid which contains one or more nucleotides which are not part of the nucleic acid template (e.g. the nucleic acid template may be of a shorter length than the primary nucleic acid which contains the nucleic acid template).

A nucleic acid template may be of any length of nucleotides. For example, a nucleic acid template may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In another example, a nucleic acid template may be between 2 and 100,000, between 5 and 100,000, between 10 and 100,000, between 15 and 100,000, between 20 and 100,000, between 25 and 100,000, between 30 and 100,000, between 50 and 100,000, between 70 and 100,000, between 100 and 100,000, between 200 and 100,000, between 2 and 10,000, between 5 and 10,000, between 10 and 10,000, between 15 and 10,000, between 20 and 10,000, between 25 and 10,000, between 30 and 10,000, between 50 and 10,000, between 70 and 10,000, between 100 and 10,000, between 200 and 10,000, between 2 and 5,000, between 5 and 5,000, between 10 and 5,000, between 15 and 5,000, between 20 and 5,000, between 25 and 5,000, between 30 and 5,000, between 50 and 5,000, between 70 and 5,000, between 100 and 5,000, between 200 and 5,000, between 2 and 3,000, between 5 and 3,000, between 10 and 3,000, between 15 and 3,000, between 20 and 3,000, between 25 and 3,000, between 30 and 3,000, between 50 and 3,000, between 70 and 3,000, between 100 and 3,000, between 200 and 3,000, between 2 and 1,000, between 5 and 1,000, between 10 and 1,000, between 15 and 1,000, between 20 and 1,000, between 25 and 1,000, between 30 and 1,000, between 50 and 1,000, between 70 and 1,000, between 100 and 1,000, between 200 and 1,000, between 2 and 500, between 5 and 500, between 10 and 500, between 15 and 500, between 20 and 500, between 25 and 500, between 30 and 500, between 50 and 500, between 70 and 500, between 100 and 500, or between 200 and 500 nucleotide bases in length.

A “primer” as used herein may refer to a polynucleotide which is i) capable of hybridizing to an original nucleic acid strand and ii) acting as a point of initiation for the synthesis of a new nucleic acid strand, wherein the new nucleic acid strand is an extension product of the primer and is complementary to the original strand. A primer may have a free —OH group at its 3′ terminus, which may serve as the origin of synthesis for the extension product.

A primer may contain standard nucleotides [e.g. standard DNA deoxyribonucleotides (deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate) or standard RNA ribonucleotides (adenosine monophosphate, guanosine monophosphate, uridine monophosphate, cytidine monophosphate)], alternative nucleotides (e.g. inosine), modified nucleotides, nucleotide analogs, or a combination thereof. For example, an oligonucleotide primer may include peptide nucleic acids, morpholinos (e.g. phosphorodiamidate morpholino oligos), locked nucleic acids [see, for example, Kaur, H, et. al, Biochemistry 45 (23), 7347-55 (2006)], glycol nucleic acids, or threose nucleic acids. A primer may have a backbone, including, for example, phosphodiester linkages, phosphorothioate linkages (a non-bridging 0 is replaced with sulfur), or peptide linkages (as part of a peptide nucleic acid). Alternative nucleotides, modified nucleotides, and nucleotide analogs may be referred to collectively herein as “non-standard nucleotides.”

The presence of a non-standard nucleotide in a primer may affect various properties of the primer. In some embodiments, inclusion of a non-standard nucleotide in a primer may increase or decrease the thermodynamic stability of a primer to a complementary sequence thereof. For example, a primer having increased thermodynamic stability may contain a locked nucleic acid. A primer having decreased thermodynamic stability may contain, for example, inosine (described by Auer et al., Nucl. Acids Res. 24; 5021-5025 (1996)) or a negatively charged chemical group, such as a carboxylic acid.

A first primer or a second primer provided herein may be of any length. The first primer and second primer may contain the same number of nucleotides, or a different number of nucleotides. In some embodiments, a first or second primer may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, a first or second primer may be no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, a first or second primer may have a length selected from a range having a minimum value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, or 1000 nucleotides in length, and a maximum value of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length.

The tail region of a first primer or a second primer provided herein may be of any length. Typically, the tail regions of a first primer and a second primer directed to the same template contain the same number of nucleotides. In some embodiments, the tail region of a first or second primer may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, the tail region of a first or second primer may be no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, the tail region of a first or second primer may have a length selected from a range having a minimum value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, or 1000 nucleotides in length, and a maximum value of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length.

The template-binding region of a first primer or a second primer provided herein may be of any length. The template-binding regions of a first primer and a second primer directed to the same template may contain the same number of nucleotides, or a different number of nucleotides. In some embodiments, the template-binding region of a first or second primer may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, the template-binding region of a first or second primer may be no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In some embodiments, the template-binding region of a first or second primer may have a length selected from a range having a minimum value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, or 1000 nucleotides in length, and a maximum value of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length.

In some embodiments, a primer may be of any length and contain any nucleotide sequence which permits sufficiently stable and specific annealing of the primer to its complement at the temperature being used for a method or step thereof involving the primer. The exact length desired of a primer may depend on a variety of factors, including the temperature of a reaction, the chemical composition of the primer, and the reaction involving the primer. In some embodiments, the template-binding region of a primer may be of any length and contain any nucleotide sequence which permits sufficiently stable and specific annealing of the template-binding region of the primer to its complement at the temperature being used for a method or step thereof involving the primer. The exact length desired of the template-binding region of a primer may depend on a variety of factors, including the temperature of a reaction, the chemical composition of the template-binding region of the primer, and the reaction involving the primer. The inclusion of one or more non-standard nucleotides in the primer may change the desired length of the primer for use in a method provided herein, as compared to the length of a corresponding primer lacking a non-standard nucleotide. For example, if with a method provided herein it is desired to have a primer with a certain melting temperature (“Tm”), in some embodiments, a primer with the selected Tm may be of a shorter length if the primer contains at least some non-standard nucleotides, as compared to if the primer contains only standard nucleotides. Generally, “melting temperature” of a nucleotide sequence refers to the temperature at which 50% of nucleic acids having the nucleotide sequence are based paired to a complementary sequence thereof (i.e. are in a double-stranded molecule), and 50% of nucleic acids having the nucleotide sequence are in single-stranded form.

In some embodiments, one or both of a first primer or second primer provided herein may be designed to contain a nucleotide sequence in the tail region of the primer which is complementary to an internal motif (i.e. a nucleotide sequence) within a target nucleic acid sequence. For example, referring again to FIG. 1, the tail region of a first primer 105 may contain a nucleotide sequence which is complementary to an internal motif in a second strand of a double-stranded nucleic acid template 104, an extension product of a first copy of the first primer 111, or an extension product of a second copy of the first primer 113. Similarly, referring still to FIG. 1, the tail region of a second primer 106 may contain a nucleotide sequence which is complementary to an internal motif in a first strand of a double-stranded nucleic acid template 103 or an extension product of the second primer 112. In at least some circumstances, having a nucleotide sequence in the tail region of a primer which is complementary to an internal motif within a target nucleic acid sequence may increase the speed or specificity of a reaction as described herein.

Without being bound by theory, it is believed that having a nucleotide sequence in the tail region of a primer which is complementary to an internal motif within a target nucleic acid sequence may increase the speed or specificity of a reaction according the mechanism outlined in FIG. 9. For brevity, FIG. 9 and this mechanism is described only with reference to a first copy of a first primer, but equally may apply to a second copy of a first primer, a second primer, or the like, as appropriate. Also, FIG. 9 and this mechanism is described only with reference to an extension product of a first copy of the first primer 111 which is annealed to an extension product of the second primer 112, but equally may apply to other molecules containing an extension product of a primer described herein. Turning now to FIG. 9, at least a portion of the tail region of a first primer may contain a nucleotide sequence 901 (checkerboard fill) which is complementary to an internal motif 902 (backslash-like fill) in an extension product of a first copy of the first primer 111 and that first primer may be integrated into the extension product of the first copy of the first primer (as described elsewhere herein) 111. The extension product of the first copy of the first primer 111 may be annealed to an extension product of the second primer 112. During the “breathing” of a duplex nucleic acid containing the extension product of the first copy of the first primer 111 and the extension product of the second primer 112, the tail region of the primer 901 may move towards and may temporarily anneal to the internal motif 902 present in the extension product of a first copy of the first primer. After temporarily annealing to the internal motif 902 in the extension product of the first copy of the first primer, the tail region of the primer 901 may move back to its original position annealed to the extension product of the second primer 112, and optionally, it may repeatedly move back and forth between these configurations (depicted by the arrows in FIG. 9). During the time that the nucleotide sequence of the tail region 901 is annealed to the internal motif 902, one or more nucleotides at the 3′ terminus of the extension product of the second primer 112 may be un-annealed, and available for binding to a new copy of a first primer or to a complementary sequence in another duplex nucleic acid (e.g. a secondary nucleic acid). In the event that one or more nucleotides at the 3′ terminus of the extension product of the second primer 112 binds to a new copy of a first primer or to a complementary sequence in another duplex nucleic acid (e.g. a secondary nucleic acid), either of these events may increase the rate of a reaction as described elsewhere herein, for example, by the generation of a new extension product of the first primer (which displaces the original first primer extension product during its generation). Also, even without there being a nucleotide sequence 901 in the first primer which is complementary to an internal motif 902, there may still be “breathing” between complementary sequences in a duplex nucleic acid in methods provided herein. However, the amount of time during which a duplex nucleic acid may “breathe” may be increased when there is a nucleotide sequence 901 in the first primer which is complementary to an internal motif 902, and this may in turn increase the rate of a reaction according to a method provided herein.

In embodiments, an internal motif may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. In embodiments, an internal motif may contain no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. In embodiments, an internal motif may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 75 and no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides (in which the upper value is above the lower value). Typically, the nucleotide sequence in the primer which is complementary to the internal motif contains the same number of nucleotides as in the internal motif. Also, the nucleotide sequence in the primer which is complementary to the internal motif may be perfectly complementary to the internal motif, or there may be one or more mismatched nucleotides between the nucleotide sequence in the primer and the internal motif. The start of the internal motif may be separated by any distance from the 3′ terminal nucleotide of the primer which is extended to form the primer extension product which contains the internal motif, but typically may be separated from the 3′ terminal nucleotide of the primer, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. In embodiments, the start of the internal motif may be separated from the 3′ terminal nucleotide of the primer which is extended to form the primer extension product which contains the internal motif by no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. In embodiments, the start of the internal motif may be separated from the 3′ terminal nucleotide of the primer which is extended to form the primer extension product which contains the internal motif by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 75 and no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides (where the upper number is greater than the lower number).

As used herein, in the context of a single target nucleic acid strand/polynucleotide template, an “internal motif” is a portion of the target nucleic acid strand to which the tail region of one primer of a primer pair to amplify the target nucleic acid provided herein is complementary, and which has the same or a similar nucleotide sequence as the tail region of the other primer of the primer pair. Thus, for example, if the internal motif in a target nucleic acid has the nucleotide sequence, in the 5′ to 3′ direction of GGGATGGA, the tail/first region of one primer of a primer pair used to amplify the target nucleic acid according to a method provided herein will have the nucleotide sequence, in the 5′ to 3′ direction of TCCATCCC (or a similar sequence), and the tail/first region of the other primer of the primer pair will have the nucleotide sequence, in the 5′ to 3′ direction of GGGATGGA (or a similar sequence). In the context of a double stranded target nucleic acid, the term “internal motif” refers to the relevant region of either of the two strands, as described above for a single strand. In the context of a primer extension product described herein, an “internal motif” refers to the portion of the extension product which is complementary to the portion of the primer extension product which corresponds to the tail region of the primer that was extended to form that particular primer extension product. Also, if reference is to be made to a nucleotide sequence in a different strand which is complementary to the “internal motif” in a particular strand, that complementary sequence may be referred to as herein as an “internal motif complement”. Of note, in situations where two complementary nucleic acid strands are present (e.g. as in a double-stranded molecule containing two complementary primer extension products), each strand will have a different “internal motif” (the “internal motif's of the different strands will be complementary to each other), so the description of an “internal motif” provided herein may be qualified with reference to the relevant strand or primer, where appropriate. For example, a double stranded nucleic acid may be described, wherein the first strand of the nucleic acid has, in 5′-3′ order, portions C′-B-C-A-C′, and the second strand of the nucleic acid has, in 5′-3′ order, portions C-A′-C′-B′-C, where portion C′ of the first strand is annealed to portion C of the second strand, portion A of the first strand is annealed to portion A′ of the second strand, etc. If portion C′ of the first strand corresponds to the tail region of a primer provided herein, and portion C′ is complementary to portion C, then portion C of the first strand may be referred to as the “internal motif” of the first strand, and portion C′ of the second strand may be referred to as the “internal motif complement” in relation to the first strand. At the same time, if portion C of the second strand also corresponds to the tail region of a primer provided here, and it is complementary to portion C′, then portion C′ of the second strand may be referred to as the “internal motif” of the second strand, and portion C of the first strand may be referred to as the “internal motif complement” in relation to the second strand.

In embodiments, methods and compositions provided herein may be described with reference to FIG. 10. Features of FIG. 10 may be described in generally the same way as FIG. 8, except that the template of FIG. 10 also includes an internal motif.

A polynucleotide template 1000 (FIG. 10A) may be a target for amplification. The polynucleotide template 1000 is a single nucleic acid strand. The polynucleotide template strand may exist as a free, single-stranded molecule, or it may exist as part of a double-stranded molecule, in which the polynucleotide template strand is annealed to a complementary strand thereof. The polynucleotide template 1000 may be DNA or RNA.

The polynucleotide template 1000 may contain at least a first portion 1001 (also indicated with an “(i)” and letter “A”) and a second portion 1002 (also indicated with an “(ii)” and letter “B”). The first portion 1001 and second portion 1002 may each contain any number of nucleotides, such as is described for the first portion and second portion of the template in FIG. 8. The first portion 1001 and second portion 1002 may contain the same or a different number of nucleotides. In addition, the polynucleotide template 1000 may contain a third portion 1003 (also indicated with an “(iii)” and letter “C”), which may also be referred to as an “internal motif” The internal motif typically contains between 6 and 10 nucleotides, although optionally may contain other numbers of nucleotides, such as at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides, no more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides and no more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (with the upper value being larger than the lower value). The internal motif 1003 typically is separated from the first portion 1001 and the second portion 1002 on either side by between 2 and 25 nucleotides, although may be separated by other numbers of nucleotides such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or 60 nucleotides, or between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or 60 nucleotides (with the upper value being larger than the lower value). In embodiments, there may be the same number of nucleotides between the first portion 1001 and the internal motif 1003, as compared to between the second portion 1002 and the internal motif 1003 (e.g. there may be 6 nucleotides between the first portion and the internal motif and 6 nucleotides between the second portion and the internal motif). In other embodiments, there may be a different number of nucleotides between the first portion 1001 and the internal motif 1003 than between the second portion 1002 and the internal motif 1003 (e.g. there may be 6 nucleotides between the first portion and the internal motif and 8 nucleotides between the second portion and the internal motif). In embodiments, there are no nucleotides between the internal motif and the first portion 1001 or the second portion 1002 (e.g. in embodiments, the internal motif may be directly next to one or both of the first portion 1001 or second portion 1002).

The polynucleotide template 1000 may be incubated in a reaction mixture containing a first primer 1010 and a second primer 1020 (FIG. 10B). Typically, the reaction mixture contains multiple copies of each primer, such as, for example, at least 4, 5, 6, 10, 15, 20, 50, 100, 1000, 10,000, 100,000, or 1 million copies or more of each of the primers. The first primer 1010 may have a first region 1011 (also indicated with an “(i)”) and a second region 1012 (also indicated with an “(ii)”). The first region of the first primer 1011 may have a 5′ end, and the second region of the first primer 1012 may have a 3′ end. The first region of the first primer may have a nucleotide sequence which is different from the nucleotide sequence of the second region of the first primer. The second primer 1020 may have a first region 1021 (also indicated with a “(i)”) and a second region 1022 (also indicated with a “(ii)”). The first region of the second primer 1021 may have a 5′ end, and the second region of the second primer 1022 may have a 3′ end. The first region of the second primer may have a nucleotide sequence which is different from the nucleotide sequence of the second region of the second primer.

The second region of the first primer 1012 may have a nucleotide sequence which is complementary to the nucleotide sequence of the first portion of the polynucleotide template 1001. In FIG. 10, nucleotide sequence of the first portion of the polynucleotide template 1001 is represented by the letter A, and the nucleotide sequence of the second region of the first primer 1012 is represented by the letter A′, where A and A′ are complementary to each other. The second region of the second primer 1022 may have a nucleotide sequence which is complementary to a partner nucleotide sequence 1030, wherein the partner nucleotide sequence 1030 is complementary to the nucleotide sequence of the second portion of the polynucleotide template 1002. In FIG. 10, the nucleotide sequence of the second portion of the polynucleotide template 1002 is represented by the letter B, the nucleotide sequence of the partner nucleotide sequence 1030 is represented by the letter B′, and the nucleotide sequence of the second region of the second primer 1022 is represented by the letter B, where B and B′ are complementary to each other. In other words, as indicated by FIG. 10, the nucleotide sequence of the second portion of the polynucleotide template 1002 (B) may be the same as the nucleotide sequence of the second region of the second primer 1022 (B). With compositions and methods provided herein, a partner nucleotide sequence 1030 may be generated, for example, as part of an extension product from the 3′ end of the first primer. In embodiments, the first region of the first primer 1011 may be complementary to the first region of the second primer 1021. In FIG. 10, the nucleotide sequence of the first region of the first primer is represented by the letter C, and the nucleotide sequence of the first region of the second primer is represented by the letter C′, where C and C′ are complementary to each other.

In addition, the first region of the second primer 1021 may be complementary to the internal motif 1003 in the polynucleotide template. In FIG. 10, the nucleotide sequence of the internal motif 1003 of the polynucleotide template is represented by the letter C, where C and C′ are complementary to each other. Thus, as indicated by FIG. 10, in embodiments, the first region of the first primer 1011 may have the same nucleotide sequence as the internal motif 1003 of the polynucleotide template, while the first region of the second primer 1021 has a sequence which is complementary to the internal motif 1003. In addition, the first region of the second primer 1021 is complementary to both the first region of the first primer 1011 and the internal motif, while the first region of the first primer 1011 is complementary to the first region of the second primer 1011 and to the internal motif complement.

In embodiments, the first primer and second primer of FIG. 10 may have any of the characteristics of the first primer and second primer described elsewhere herein (e.g. as in FIG. 1 or 8), respectively. Also, in embodiments, the first region of the first primer of FIG. 10 may have any of the characteristics of the tail region of a first primer described elsewhere herein. In embodiments, the second region of the first primer of FIG. 10 may have any of the characteristics of the template binding region of a first primer described elsewhere herein. In embodiments, the first region of the second primer of FIG. 10 may have any of the characteristics of the tail region of a second primer described elsewhere herein. In embodiments, the second region of the second primer of FIG. 10 may have any of the characteristics of the template binding region of a second primer described elsewhere herein.

In embodiments, a reaction mixture containing a polynucleotide template 1000 and at least two copies of a first primer 1010 and at least two copies of a second primer 1020 may contain one or more other components which may support polymerase-based nucleic acid synthesis as described elsewhere herein (e.g. polymerases, nucleotides, buffers, water, etc.). Similarly, a reaction mixture containing a polynucleotide template 1000 and at least two copies of a first primer 1010 and at least two copies of a second primer 1020 may be incubated at any of the reaction conditions described elsewhere herein for supporting the amplification of a nucleic acid template or the generation of concatemers.

In embodiments, upon incubation of a polynucleotide template 1000 and at least two copies of a first primer 1010 and at least two copies of a second primer 1020 in a reaction mixture as described herein, one or more concatemer strands may be formed (FIG. 10C). In embodiments, a formed concatemer strand 1040 may have a 5′ end and a 3′ end, and may have a sequence containing the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein C′ is the nucleotide sequence of the first region of the second primer, T is the sequence of the polynucleotide template or an analogous sequence thereof, and X is any number and sequence of nucleotides. As further indicated by FIG. 10C, within the sequence of the polynucleotide template of strand 1040, in the 5′ to 3′ direction, the sequence of the following portions of the template are present: B (the second portion of the polynucleotide template), C (the internal motif), and A (the first portion of the polynucleotide template). Thus, as shown in FIG. 10C, the sequence of the internal motif/internal motif complement occurs at multiple positions in a concatemer strand in embodiments provided herein. Specifically, in strand 1040, the sequence of the internal motif complement (C′) appears in the concatemer strand between the two copies of the sequence of the polynucleotide template and at the ends of the strand, and the sequence of the internal motif (C) occurs in the same strand within the sequence of the polynucleotide template. Thus, as indicated by the general structure of the concatemer strand 1040, a concatemer strand may contain at least two copies of the sequence of the polynucleotide template or an analogous sequence thereof, where the copies of the sequence of the polynucleotide template are separated by at least the sequence of the first region of the second primer. In embodiments, T in the general structure C′-T-C′-T-X-C′ provided above may be an analogous sequence of the polynucleotide template.

In embodiments, a concatemer strand 1040 generated according to method provided herein may be part of a single-stranded molecule. In other embodiments, a concatemer strand 1040 generated according to method provided herein may be part of a double-stranded concatemer, in which the concatemer strand 1040 is annealed to a strand which is complementary to the concatemer strand 1050. While FIG. 10C depicts concatemer strands having a sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′ (strand 1040) or C-T′-C-T′-X′-C (strand 1050), wherein X is 0 nucleotides, a concatemer strand generated according to the schematic of FIG. 10 may have any of the characteristics of a concatemer strand described above or elsewhere herein. Also, as shown in FIG. 10C, sequences A and B and C are part of the template strand, and thus are not separately notated in the general structure C′-T-C′-T-X-C′ or C-T′-C-T′-X′-C.

To further indicate the relationship of elements in embodiments of FIG. 10, blocks 1060, 1062, 1064, 1066, and 1068 are provided in FIG. 10. Block 1060 (with a backslash-like fill) indicates the relative position of the total polynucleotide template sequence (i.e. element 1000), or its complementary sequence. Block 1062 (with vertical line fill) indicates the relative position of the sequence of first portion of the polynucleotide template (i.e. the relative position of the sequence of element 1001), or its complementary sequence. Thus, block 1062 also represents the sequence of the second region of the first primer (i.e. element 1012), which is complementary to element 1001. Block 1064 (with vertical and horizontal line fill) indicates the relative position of the sequence of the second portion of the polynucleotide template (i.e. the relative position of the sequence of element 1002), or its complementary sequence. Thus, block 1064 also represents the sequence of the partner nucleotide sequence (i.e. element 1030), which is complementary to element 1002. Block 1064 also indicates the relative position of the sequence of the second region of the second primer 1022, which may be the same sequence as the sequence of the second portion of the polynucleotide template (element 1002), or a similar sequence. In other words, in embodiments the nucleotide sequence of 1022 may be the same as or similar to nucleotide sequence of 1002. As depicted, for example, in FIG. 10A, in embodiments, there are nucleotides in the polynucleotide template which are not part of the first portion, second portion, or third portion (internal motif) of the polynucleotide template. Block 1066 (with wavy line fill) indicates the relative position of the sequence of the first region of the first primer (i.e. the relative position of the sequence of element 1011/region “C”), or its complementary sequence. Thus, block 1066 also represents the sequence of the first region of the second primer (element 1021/region “C”), which is complementary to element 1011. In addition, block 1066 also represents the sequence of the internal motif or its complementary sequence. Of note, the sequence of the internal motif or its complementary sequence is both in the template sequence, as well as in the tails of the first and second primers. As described elsewhere herein, the internal motif may be directly adjacent to one or both of the first portion or the second portion of the polynucleotide template, or there may be additional nucleotides between the internal motif and the first portion or the second portion. Block 1068 (with diamond fill) indicates the relative position of the sequence of the concatemer strand (element 1040), or its complementary sequence. Thus, block 1068 also represents the sequence of a strand which is complementary to the concatemer strand (element 1050). As depicted in FIG. 10C, within the concatemer strand or its complement (1068), two copies of the polynucleotide template sequence or its complement (1060) are present, separated by the nucleotide sequence of the first region of the first primer or its complement (1066). This sequence separating the copies of the polynucleotide template is also the sequence of the internal motif or its complement (1066). In addition, within the concatemer strand or its complement (1068), the nucleotide sequence of the first region of the first primer or its complement (1066) flank the polynucleotide template sequence or its complement (1060) on both ends. Also, within the concatemer strand or its complement (1068), there are two copies of the sequence of the second region of the first primer or its complement (1062) and two copies of the sequence of the sequence of the second region of the second primer or its complement (1064), which are both present in two copies as part of the two copies of the polynucleotide template sequence or its complement. Blocks 1060, 1062, 1064, 1066, and 1068 are not to be interpreted to as to dictate any particular required size relationship between the elements of FIG. 10; rather blocks 1060, 1062, 1064, 1066, and 1068 are simply to help indicate the general position of the elements of FIG. 10 relative to each other in certain embodiments provided herein.

A primer provided herein may be prepared by any suitable method. For example, a primer may be chemically synthesized. In another example, a naturally occurring nucleic acid may be isolated, cleaved (e.g. with restriction enzymes), and/or modified to generate or to become part of a primer described herein.

In some embodiments, a label may be attached to a primer. Labels include, for example, binding ligands (e.g. digoxin or biotin), enzymes, fluorescent molecules/fluorophores, luminescent molecules, quencher molecules, or radioisotopes. In other embodiments, a base of an oligonucleotide may be replaced with a fluorescent analog, such as 2-aminopurine (see, for example, Proc. Acad. Sci. USA, 91, 6644-6648 (1994), which is herein incorporated by reference in its entirety).

In some embodiments, conditions such that: i) a template-binding region of a first copy of a first primer anneals to a strand of a nucleic acid template, ii) a template-binding region of a second primer anneals to an extension product of a first copy of a first primer, iii) a template-binding region of a second copy of a first primer anneals to an extension product of a second primer, or iv) a 3′ terminal region of an extension product of a second copy of a first primer of a first copy of a secondary nucleic acid anneals to a 3′ terminal region of an extension product of a second primer of a second copy of a secondary nucleic acid, to produce a cross-over structure comprising these strands, may each include (i.e. any of i), ii), iii), or iv) may include) incubating the nucleic acids at a temperature such that the strands of double-stranded nucleic acid molecules “breathe” (i.e. undergo brief periods of localized rupture of hydrogen bonds connecting base pairs) to a degree sufficient to facilitate the entry of a primer or different nucleic acid strand between the strands of a double-stranded molecule, and sufficient to permit the annealing of the primer or different nucleic acid strand to one of the strands of the opened double-stranded nucleic acid molecule. In some embodiments, methods or steps thereof may be performed or incubated at a temperature of at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, or 95 C. In some embodiments, methods or steps thereof may be performed or incubated at a temperature of no greater than 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, or 95 C. In some embodiments, methods or steps thereof may be performed or incubated at a temperature between 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, or 90 C and 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90 or 95 C.

In some embodiments, for a method or steps thereof provided herein, the step or method is performed at a temperature below the melting temperature (Tm) of the relevant potentially paired nucleotide strands, or regions thereof (e.g. the template-binding region of the first primer to the first strand of a nucleic acid template, the template-binding region of the second primer to the extension product of the first copy of the first primer, the first primer to the extension product of the second primer, the 3′ terminal region of the extension product of the second primer to its complement, the 3′ terminal region of the extension product of the first copy of the first primer to its complement, etc.). In some embodiments, for a method or steps thereof provided herein, the step or method is performed at a temperature above the Tm of the relevant potentially paired nucleotide strands, or regions thereof (e.g. the template-binding region of the first primer to the first strand of a nucleic acid template, the template-binding region of the second primer to the extension product of the first copy of the first primer, the first primer to the extension product of the second primer, the 3′ terminal region of the extension product of the second primer to its complement, the 3′ terminal region of the extension product of the first copy of the first primer to its complement, etc.). In some embodiments, for a method or steps thereof provided herein, the step or method is performed a ta temperature at or within +/−1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 C of the Tm of the relevant potentially paired nucleotide strands, or regions thereof (e.g. the template-binding region of the first primer to the first strand of a nucleic acid template, the template-binding region of the second primer to the extension product of the first copy of the first primer, the first primer to the extension product of the second primer, the 3′ terminal region of the extension product of the second primer to its complement, the 3′ terminal region of the extension product of the first copy of the first primer to its complement, etc.).

In some embodiments, a nucleic acid polymerase is included with a method or composition provided herein. A polymerase may generate an extension product of a primer. The primer and extension product thereof may be complementary to a template nucleic acid strand. Generally, a nucleic acid polymerase will initiate synthesis of an extension product of a primer at the 3′ end of the primer. In some embodiments, a DNA polymerase is included with a method or composition provided herein. As used herein, a “DNA polymerase” refers to a nucleic acid polymerase which has primary or exclusive polymerase activity on DNA templates. In some embodiments, a reverse transcriptase is included with a method or composition provided herein. As used herein, a “reverse transcriptase” refers to a nucleic acid polymerase which can synthesize a DNA strand from an RNA template. In some embodiments, an RNA polymerase may be included with a method or composition provided herein. As used herein, a “RNA polymerase” refers to a nucleic acid polymerase which can synthesize an RNA strand from a DNA or RNA template.

In some embodiments, a polymerase provided herein may have strand displacement activity. Polymerases having strand displacement activity include, for example, exo-Bca DNA polymerase, phi29 DNA polymerase, Klenow Fragment of E. coli DNA Polymerase I, VentR DNA polymerase, Deep VentR DNA polymerase, 9° Nm DNA polymerase, Bst 2.0 DNA polymerase, and Large Fragment of Bst DNA Polymerase. Other polymerases having strand displacement activity may also be used.

Modified versions of polymerases may also be used with the methods and compositions provided herein, provided that the modified polymerase has sequence-dependent nucleic acid synthesis activity. A modified version of a polymerase (“modified polymerase”) may have, for example, 100 or fewer, 70 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 different amino acid from the sequence of the parent version of the polymerase. In some embodiments, a modified polymerase may contain no more than 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, or 5 greater or fewer amino acids than the parent polymerase. In some embodiments, a modified polymerase may comprise a fragment of a parent polymerase. In some embodiments, a modified polymerase may comprise a chimeric polypeptide with a portion derived from a polymerase and a portion derived from a non-polymerase protein. In some embodiments, a modified polymerase may have, for example, increased catalytic activity, increased stability, or increased thermostability as compared to the parent polymerase.

In some embodiments, a polymerase provided herein is thermostable. A thermostable polymerase may have, for example, a half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes at a temperature of up to 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 C. In some embodiments, a modified polymerase may be thermostable.

In some embodiments, methods and steps thereof provided herein include or are performed under conditions sufficient to support polymerase-based nucleic acid synthesis. Example conditions for polymerase-based nucleic acid synthesis are known in the art and are provided, for example, in Green and Sambrook, supra. Non-limiting components for a polymerase-based nucleic acid synthesis reaction may include one or more of: polymerase enzyme (at a concentration between, for example, 0.01 and 10 units enzyme per 50 microliters reaction volume, or any range therein including, for example, between 0.01-1, 0.1-10, 0.1-5, 0.5-10, 0.5-5, 0.5-2, 1-10, or 1-5 units enzyme per 50 microliters reaction volume, where 1 unit of enzyme will incorporate 15 nmol of dNTPs into polymerization product in 30 minutes at 75 C); template (at a concentration of at least, for example, 1, 10, 100, 1,000, 10,000, or 100,000 copies per reaction); primer (at a concentration between, for example, 0.01 and 10 micromolar, or any range therein including, for example, between 0.01-1, 0.1-10, 0.1-5, 0.5-5, or 0.5-2 micromolar); dNTPs (e.g. dATP, dTTP, dGTP, and dCTP, at a concentration between, for example, 50 and 500 micromolar each of dATP, dTTP, dGTP, and dCTP, or any range therein including, for example, between 50-350, 100-500, 100-300, 200-500, or 300-400 micromolar each of dATP, dTTP, dGTP, and dCTP); salt (e.g. KCl or potassium acetate, at a concentration between, for example, 1 and 200 millimolar, or any range therein including, for example, between 1-100, 1-50, 1-20, 1-10, 10-20, 10-50, or 10-200 millimolar); buffer (e.g. Tris-HCl or Tris-acetate, pH 7.8-8.5, at a concentration between, for example, 1 and 100 millimolar, or any range therein including, for example, between 1-50, 1-20, 1-10, 1-5, 10-100, 20-100, or 50-100 millimolar); and magnesium ions (at a concentration between, for example 0.1 and 10 millimolar, or any range therein, including, for example, between 0.1-5, 0.1-1, 0.5-10, 0.5-5, or 0.5-2.5 millimolar). Additional non-limiting components for a polymerase-based nucleic acid synthesis reaction may increase the speed of the reaction, increase the fidelity of the reaction, or increase the stability of enzymes or DNA in the reaction, and may include one or more of: gelatin (at a concentration between, for example, 0.0001% and 0.1% w/v), BSA (at a concentration between, for example, 0.01 and 1 microgram per microliter), sucrose (at a concentration between, for example 0.01 molar and 0.8 molar), trehalose (at a concentration between, for example 0.01 molar and 0.8 molar), DMSO (at a concentration between, for example, 0.01 and 10% v/v), betaine (at a concentration between, for example, 0.1 and 10 molar), formamide (at a concentration between, for example, 0.1 and 10% v/v), glycerol (at a concentration between, for example, 0.1 and 20% v/v), polyethylene glycol (at a concentration between, for example, 0.1 and 20% v/v), non-ionic detergents [e.g. NP-40 (at a concentration between, for example, 0.01 and 1% v/v), Tween-20 (at a concentration between, for example, 0.01 and 1% v/v), or Triton X-100 (at a concentration between, for example, 0.01 and 1% v/v)], ammonium ions [e.g. ammonium sulfate (at a concentration between, for example, 1 and 100 millimolar)], and EDTA (at a concentration between, for example, 0.001 and 0.1 millimolar). Other reagents may also be present in a polymerase-based nucleic acid synthesis reaction provided herein. For example, reagents sufficient to synthesize RNA reaction products or reaction products containing non-standard nucleotides may be used. Conditions sufficient to support polymerase-based nucleic acid synthesis may include a variety of temperatures and pH values. For example, the pH of a polymerase-based nucleic acid synthesis reaction may be between, for example pH 6.0 and pH 10.0, such as 6.5, 7, 7.5, 7.8, 7.9, 8, 8.1, 8.2, 8.5, 9, or 9.5. The temperature of a polymerase-based nucleic acid synthesis reaction may be constant or varied. A constant temperature may be between, for example, 10 C and 95 C, such as 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, or 85 C. A varied temperature may be two or more different temperatures between, for example, 10 C and 95 C, such as two or more temperatures selected from 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, or 85 C.

Methods provided herein may be performed at a variety of temperatures. In some embodiments, all steps of a method are performed at the same temperature. Thus, temperature cycling such as in PCR is not necessary with methods disclosed herein. In some embodiments, methods provided herein may be performed at two or more different temperatures. In some embodiments, a reaction mixture containing reagents for a method provided herein is incubated at two or more different temperatures. In some examples, different temperatures may be selected to optimize the rate, accuracy, or other feature of different steps of a method provided herein. For example, a temperature may be selected to increase the enzymatic activity of a polymerase. In some examples, different temperatures may be selected to increase the binding specificity of a primer to a template or to increase the accessibility of a template to a primer (e.g. higher temperatures may promote the separation of duplex template nucleic acids). In some embodiments, all of the steps of a method provided herein are performed at a temperature of no greater than 80, 70, 60, 50, 40, 30, 20 or 10° C. In some embodiments, a method provided herein is performed at a temperature between 20-60, 30-70, 40-80, 20-40, 30-50, 40-60, 50-70, 60-80, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65° C. In certain embodiments, a sample containing a target nucleic acid may be heated to a temperature greater than 40, 50, 60, 70, 80, 90, or 95 C before the initiation of a method provided herein. In certain embodiments, a reaction mixture provided herein may be heated one time to an elevated temperature greater than 40, 50, 60, 70, 75, 80, 85, 90, or 95 C before or after the initiation of a method provided herein. After heating the reaction mixture to the elevated temperature, it may be maintained at a lower temperature as provided elsewhere herein (e.g. at a temperature between 40-70 C) for the remainder of the performance of the method. In embodiments, if a reaction mixture or sample is heated to an elevated temperature before the initiation of a method provided herein, a nucleic acid polymerase may be added to the reaction mixture or sample after the reaction mixture or sample has been heated to the elevated temperature, and the reaction mixture or sample has been returned to a lower temperature as provided herein. Methods disclosed herein may be performed with or without a thermocycler.

As one consideration, the temperature used for a method or step thereof provided herein may be selected to be appropriate for the enzyme(s) being used in the step of the method. In some embodiments, for methods in which a polymerase is used, the temperature(s) of the reaction is selected such that it does not significantly impair the activity of the polymerase (e.g. the temperature of the reaction may be selected such that polymerase has a half-life of at least 24, 12, 6, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 hours). Alternatively, methods may be performed at a temperature that impairs the activity of the enzyme(s) being used in the method (e.g. the temperature of the reaction may be selected such that an enzyme in the reaction has a half-life of no more than 24, 12, 6, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 hours). In some embodiments, if a method is performed at a temperature or other condition (e.g. pH) that impairs the activity of one or more enzyme(s), additional enzyme may be added to the reaction at one or more intervals after the initiation of the method to supplement the activity of the impaired enzyme(s).

In some embodiments, one or more steps of a method provided herein occur in the same reaction vessel (e.g. tube, tip, container, etc.). In some embodiments, all of the steps of a method occur in the same reaction vessel.

Reagents for methods provided herein can all be provided together at the start of a reaction, or they may be added sequentially, where after one, two, or more steps new reagents are added to a reaction. In some circumstances, new reagents (e.g. enzymes, primers) may be added to a reaction vessel during the course of the reaction, to increase the amount of reagents available to act on substrates or to replace the function of reagents that have become inactivated (e.g. enzymes). New reagents may be added to a reaction at one or more selected time intervals after the initiation of a reaction of a method provided herein (for example, at 1, 3, 5, 7, 10, 15, 20, 30, 45, or 60 minutes after the initiation of a reaction).

In some embodiments, one or more steps of a method provided herein may occur simultaneously. For example, after the generation of a single copy of an extension product of a first copy of the first primer, multiple copies of an extension product of a second primer may be sequentially generated from that single copy of an extension product of a first copy of the first primer. As copies of the extension product of a second primer are sequentially generated from the single copy of the extension product of a first copy of the first primer, the individual copies of the extension product of a second primer may, for example, serve as a template for the formation of an extension product of a second copy of the first primer, be a component of a secondary nucleic acid, be a component of a cross-over structure, or may be an initiation point for the formation of an extension product of an extension product of a second primer/first concatemer strand. In another example, two copies of a secondary nucleic acid may form a cross-over structure at the same time that an extension product of a first copy of a first primer is generated from a first strand of a nucleic acid template. In another example, one copy of an extension product of a first copy of a first primer is generated from a first strand of a nucleic acid template at the same time that an extension product of the second primer is generated from a different copy of the extension product of a first copy of a first primer. Other steps provided herein may also occur simultaneously. In addition, in methods and compositions provided herein involving a double-stranded nucleic acid template, either strand of the nucleic acid template may be considered a “first strand”, and either primer of a primer pair provided herein may be considered a “first primer”. Accordingly, in some examples, both strands of a double-stranded nucleic acid may be simultaneously used as “first strand” of a nucleic acid according to a method provided herein, with opposite primers of the primer pair serving as the “first primer” for the different “first strands”. Also, various structures generated according to methods provided herein may optionally enter different pathways of methods provided herein. For example, in embodiments, a first secondary nucleic acid may pair with a second secondary nucleic acid to form a cross-over structure as described elsewhere herein. In other embodiments, a first secondary nucleic acid may be invaded by a first primer, and the extension product of the second primer may serve as a template for the generation of a new extension product of the first primer. In order to generate the new extension product of the first primer, the extension product of the first primer which is part of the secondary nucleic acid is displaced. In another example, in embodiments, a first concatemer may pair with another concatemer to form a cross-over structure. In other embodiments, a first concatemer may be invaded by a first primer, and the first concatemer strand may serve as a template for the generation of a new second concatemer strand. In order to generate the new first concatemer strand, the first concatemer strand which is part of the concatemer is displaced. Other similar events may also occur during methods provided herein.

Reactions and compositions provided herein may contain multiple copies of first primers, second primers, primary nucleic acids, secondary nucleic acids, extension products of a first copy of the first primer, extension products of a second copy of the first primer, extension products of the second primer, cross-over structures, concatemers, and the like. Accordingly, methods provided herein may include processes wherein multiple steps provided herein occur simultaneously, and such steps may occur with multiple copies of the relevant molecules.

In some embodiments, two or more sets of first and second primers are provided in a method or composition provided herein, where each set contains a first primer and a second primer, and where different primer sets are complementary to different nucleic acid templates. The template-binding region of both the first and second primers in a set may be complementary to different strands of the same double-stranded nucleic acid template. Typically, the tail regions of the first and second primers in a given set have different, non-complementary sequences from the tail regions of the first and second primer of other primer sets to be used in the same method or composition, so that the primer tails from the different primer sets are not complementary. This may be desirable in order to prevent the formation of hybrid cross-over structures containing strands derived from different nucleic acid templates. Alternatively, tail regions of the first and second primers in two or more different primer sets may have complementary sequences, in order to create hybrid cross-over structures and concatemers containing strands derived from different nucleic acid templates. Inclusion of two or more primer sets in a method or composition provided herein may support the simultaneous amplification of multiple different nucleic acid templates in the same reaction vessel. This may be useful, for example, for amplifying multiple templates of interest in a sample, or for assaying for the presence of multiple different templates in a sample. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 100, 200, 500 or more sets of first and second primers are provided in a method provided herein, in order to amplify or assay for the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 100, 200, 500 or more different nucleic acid templates.

In some embodiments, in a method provided herein, a nucleic acid template may be amplified rapidly. For example, in some embodiments, a nucleic acid template may be amplified at least 500-fold within 0.1, 0.5, 1, 3, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes of starting the method. In another example, in some embodiments, a nucleic acid template may be amplified at least 10,000-fold within 0.1, 0.5, 1, 3, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes of starting the method. In another example, in some embodiments, a nucleic acid template may be amplified at least 5, 10, 25, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000-fold over the original amount of the nucleic acid template present in a reaction mixture at the start of the method within 0.1 minute, 0.5 minute, 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours of initiation of the method. In some embodiments, when a method is initiated, all of the reagents for the first step of the method are in a vessel containing the reaction mixture for the method. In some embodiments, when a method is initiated, all of the reagents for all of the steps of the method are in a vessel containing the reaction mixture for the method.

In some embodiments, in a method provided herein, a nucleic acid template may be amplified at greater than a linear rate. In some embodiments, in a method provided herein, a nucleic acid template may be amplified exponentially. In some embodiments, in a method provided herein, a nucleic acid template may at least double in number every 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or 240 minutes after the initiation of the method. In some embodiments, a nucleic acid template may amplified at least 5, 10, 25, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or 10,000,000-fold over the original amount of the nucleic acid template present in the reaction at the start of the method.

The presence of multiple copies of a nucleic acid template in a concatemer generated according to a method provided herein may contribute to the rapid amplification of nucleic acid templates according to methods provided herein. In particular, since multiple copies of a strand of a nucleic acid template may be present in a single concatemer strand, the loading of a single polymerase onto a single concatemer strand may result in the generation of multiple copies of a strand of the nucleic acid template as the polymerase moves along the concatemer strand. In some situations, the time required for nucleic acid polymerases to encounter and load onto nucleic acid strands may significantly impact the overall speed of an amplification reaction. For example, if each nucleic acid strand that a polymerase encounters during a replication reaction only contains a single copy of a strand of a nucleic acid template, a polymerase may need to encounter and load onto a new template strand after each copy of the strand of the template is generated. In contrast, with a concatemer, after the polymerase encounters and loads on a concatemer strand, it may synthesize multiple copies of a strand of the template without needing to leave the concatemer strand or encounter and load onto another strand.

In some embodiments, provided herein are methods and compositions for the generation of a double-stranded DNA concatemer from a single-stranded or double-stranded RNA template. The method may be performed as described herein, except that a reverse transcriptase enzyme (e.g. AMV reverse transcriptase, M-MLV reverse transcriptase, Superscript II™ reverse transcriptase, Superscript III™ reverse transcriptase, or ThermoScript™ reverse transcriptase) is also included with the methods provided herein. The first copy of the first primer may anneal to the RNA template, and the reverse transcriptase enzyme may generate the extension product of the first copy of the first primer, which is formed as a DNA strand by the process of reverse transcription. Methods and conditions for reverse transcription of RNA are known in the art and are disclosed, for example, in RNA: A Laboratory Manual, D. Rio et al., Cold Spring Harbor Laboratory Press (2011), which is herein incorporated by reference in its entirety. After the generation (from an RNA strand) of the extension product of the first copy of the first primer (which is a DNA strand) by a reverse transcriptase enzyme, the rest of the steps for the generation of a concatemer may be the same as described elsewhere herein.

In some embodiments, methods and compositions provided herein may include one or more “bumping primers”. Bumping primers may be used with methods and compositions provided herein to, for example, increase the rate or specificity of generation of reaction products by, for example, increasing the rate at which a second strand of nucleic acid template is displaced from a first strand of nucleic acid template, increasing the rate at which a first strand of a nucleic acid template is displaced from a first primer extension product, or increasing the rate at which a first primer extension product is displaced from a second primer extension product. As used herein a “bumping primer” refers to a primer which is complementary to a sequence on a first strand or second strand of a nucleic acid template which is downstream from a sequence on the same strand to which the template-binding region of a first primer or second primer binds. Thus, when a bumping primer is annealed to the same nucleic acid strand that a first primer or second primer is annealed to, the 3′ terminus of the bumping primer is oriented towards the 5′ terminus of the first primer or second primer (i.e. the 5′ terminal nucleotide and tail region of the first primer or second primer). When incubated with a nucleic acid polymerase and under conditions to support the generation of primer extension products, an extension product of a bumping primer may be formed by the polymerase, from the 3′ terminus of the bumping primer. Since the 3′ terminus of the bumping primer is oriented toward the 5′ terminus of the first primer or second primer, as the extension product of the bumping primer increases in length, it may eventually encounter the 5′ terminus of the first primer or second primer as the extension product. The polymerase may then displace the first primer or second primer from the strand, as well as any extension product of the first primer or second primer. Accordingly, bumping primers may accelerate reactions provided herein.

In some embodiments, provided herein is a vessel containing one or more enzymes, primers, or other reagents provided herein. Vessels may include any structure capable of supporting or containing a liquid or solid material and may include, tubes, containers, tips, etc. In some embodiments, a wall of a vessel may permit the transmission of light through the wall. A vessel may be optically clear. A vessel may contain, for example, any one or more of an isolated nucleic acid polymerase, an isolated DNA polymerase, an isolated reverse transcriptase, a first primer, a second primer, a nucleic acid dye, or a nucleic acid probe, as described elsewhere herein. Any number of copies of any of the contents of a vessel may be provided (e.g. a first copy, a second copy, a third copy, etc.) The contents of a vessel may be in fluid communication. In some embodiments, a vessel may further contain a nucleic acid template. In some embodiments, a vessel may further contain nucleotides, buffers, salts, water, or other reagents provided herein for the amplification of nucleic acids. In some embodiments, a vessel may contain two or more sets of primers, wherein each primer set comprises a first and second primer, and the different primer sets are complementary to different nucleic acid templates.

Two or more reagents useful for a method provided herein may be packaged and provided as a kit. For example, a kit may include any two or more of: a nucleic acid template, a first primer, a second primer, a nucleic acid polymerase, a DNA polymerase, a reverse transcriptase, buffers, a nucleic acid dyes, a nucleic acid probe, or dNTPs, as described elsewhere herein. Within the kit, the two or more reagents may be packaged in separate vessels or the same vessel. In some embodiments, a kit may further contain nucleotides, buffers, salts, water, or other reagents provided herein for the amplification of nucleic acids.

In embodiments, a first primer and second primer as provided herein may be provided together as a primer set. The primer set may be provided as a stand-alone kit or composition, or the primer set may be provided in a kit with one or more other reagents for performing a method provided herein.

In some embodiments, a nucleic acid ligase may be included with a method or composition provided herein. Ligases catalyze the formation of phosphodiester bonds between nucleotides, typically between the 5′ phosphate of one nucleotide, and the 3′ hydroxyl group of another nucleotide. A reaction provided herein may amplify a target nucleic acid at a greater rate with the inclusion of a ligase in the reaction, as compared to the reaction without the inclusion of a ligase. A ligase may, for example, increase the size or number of concatemers present in a reaction provided herein.

Nucleic acid ligases include E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Ampligase™, T4 RNA ligase 1, and T4 RNA ligase 2.

In order to catalyze the ligation reaction, certain ligases require ATP (e.g. T4 DNA ligase) or NAD+(E. coli DNA ligase). In some embodiments, a ligase may ligate nucleic acids having blunt ends. In some embodiments, a ligase may ligate nucleic acids having sticky ends. In some embodiments, a ligase may ligate nucleic acids having both blunt and sticky ends.

Modified versions ligases may also be used with the methods and compositions provided herein, provided that the modified ligase has the ability to catalyze the formation of phosphodiester bonds between nucleotides. A modified version of a ligase (“modified ligase”) may have, for example, 100 or fewer, 70 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 different amino acid from the sequence of the parent, naturally occurring version of the ligase. In some embodiments, a modified ligase may contain no more than 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, or 5 greater or fewer amino acids than the parent ligase. In some embodiments, a modified ligase may comprise a fragment of a parent ligase. In some embodiments, a modified ligase may comprise a chimeric polypeptide with a portion derived from a ligase and a portion derived from a non-ligase protein. In some embodiments, a modified ligase may have, for example, increased catalytic activity, increased stability, or increased thermostability as compared to the parent ligase.

In some embodiments, a ligase provided herein is thermostable. A thermostable ligase may have, for example, a half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes at a temperature of at up to 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 C. In some embodiments, a modified ligase may be thermostable.

In some embodiments, a ligase included with methods and compositions provided herein may be a modified ligase referred to herein as “p50-Tth”, which has the amino acid sentience:

(SEQ ID NO: 79) MGHHHHHHHHHHSSGHIEGRASADGPYLQILEQPKQRGFRFRYVCEGPSH GGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGK HCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEAKIITEACIRGY NPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLM FTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGG EHYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTP KYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKG SSGTSGGGSGGGMTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYD RLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNA FNLDELKAFEERIERALGRKGPFAYTVEHKVDGLSVNLYYEEGVLVYGAT RGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEVYMPIEAFLRLNEE LEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFYALGLGLEEVERE GVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEA DGVVVKLDELALWRELGYTARAPRFAIAYKFPAEEKETRLLDVVFQVGRT GRVTPVGILEPVFLEGSEVSRVTLHNESYIEELDIRIGDWVLVHKAGGVI PEVLRVLKERRTGEERPIRWPETCPECGHRLLKEGKVHRCPNPLCPAKRF EAIRHFASRKAMDIQGLGEKLIERLLEKGLVKDVADLYRLRKEDLVGLER MGEKSAQNLLRQIEESKKRGLERLLYALGLPGVGEVLARNLAARFGNMDR LLEASLEELLEVEEVGELTARAILETLKDPAFRDLVRRLKEAGVEMEAKE KGGEALKGLTFVITGELSRPREEVKALLRRLGAKVTDSVSRKTSYLVVGE NPGSKLEKARALGVPTLTEEELYRLLEARTGKKAEELV. Ligase p50-Tth has thermostable blunt-end ligation activity at temperatures of at least 60 C. Ligase p50-Tth is a chimeric protein which comprises a His10-containing leader sequence (“His10” disclosed as SEQ ID NO: 112), a p50 sequence from the human NF-kappa-B protein accession number NP 003989 amino acids 40-366 (indicated in italics), a flexible glycine rich sequence, and a Tth DNA ligase sequence, from Thermus thermophilus HB8, accession YP_144363 (indicated with underlining). In some embodiments, a modified version of p50-Tth ligase may be used with methods and compositions provided herein (e.g. with 100 or fewer, 70 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 different amino acids from p50-Tth ligase). In embodiments, a ligase used with a composition or method provided herein may be a ligase described in U.S. Provisional Patent Application No. 61/802,124, filed Mar. 15, 2013 or PCT Application No. PCT/US14/30003, filed Mar. 15, 2014, both of which are herein incorporated by reference in their entirety for all purposes.

The various methods and compositions provided herein for the amplification of a nucleic acid template/the generation of a concatemer containing at least two copies of the template can fulfill many of the functions that have previously been carried out by other methods and compositions for isothermal and thermocycler-dependent nucleic acid amplification. A nucleic acid template for amplification according to methods provided herein may also be referred to herein as a “target nucleic acid” or the like. Methods and compositions provided herein may be used, for example, for isolation and cloning of nucleic acids of interest, gene expression analysis, diagnostic identification of nucleic acids, synthesis of novel nucleic acids, nucleic acid probe synthesis and labeling, forensic identification of a subject, allele identification from a subject, genetic screening, nucleic acid sequencing, and related applications. A target nucleic acid molecule may be of any type, including single-stranded or double stranded and DNA or RNA (e.g. mRNA). A target nucleic acid may be of any type or function (e.g. a protein-coding sequence, a regulatory sequence, an intron, etc.). A target nucleic acid may be the entirety of a gene, or a portion thereof.

In some embodiments, a method or composition provided herein may be used to detect the amount of a target nucleic acid in a sample (including the presence or absence of the target), to measure the amount of an amplification product of a target formed from a sample in a selected period of time, or to determine the amount of time necessary to generate a certain number of copies of a template from a sample. Samples which may be used with methods and compositions provided herein are described elsewhere herein, and may include, for example, a bodily fluid, a secretion, or a tissue of a subject. In embodiments, a sample may be processed prior to use of the sample in an assay to amplify a target nucleic acid in the sample according to a method provided herein. Processing of the sample may include any processing step as described elsewhere herein, and may include, for example, sonication or chemical lysing steps.

In some embodiments, a method provided herein may be performed to simultaneously assay for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more different target nucleic acids in the same reaction vessel. Typically, for each target nucleic acid of interest, a first primer and a second primer are provided, each being complementary to a strand of the nucleic acid target, or a complement thereof. The amplification of the different target nucleic acids in the same vessel may be monitored, for example, by the use of nucleic acid probes having sequence specificity for detection sequences in the different target nucleic acids, and different fluorophores.

In some embodiments, a method or composition provided herein may be used to detect the presence or absence of a particular nucleotide of interest in a target nucleic acid (e.g. in the case of a mutation or SNP). For example, a first or second primer may be selected which selectively binds to a region in a target nucleic acid which includes or is adjacent to the nucleotide of interest. The primer may be designed such that it selectively either: i) binds to the region when the region contains the nucleotide of interest, or ii) does not bind to the region when the region contains the nucleotide of interest. A method as described herein may be performed with the selected primer, and the outcome of the amplification reaction may provide information regarding the presence or absence of the nucleotide of interest in the target nucleic acid. For example, if the template-binding region of a first primer is designed to have a nucleotide sequence which is complementary to a sequence in the target nucleic acid which includes a particular nucleotide of interest (e.g. a mutation), successful amplification of the target nucleic acid with the selected primer from a sample may indicate that the sample contains a target nucleic acid having the particular nucleotide of interest. In some embodiments, a primer used for analysis of a nucleotide of interest in a target nucleic acid may contain a critical nucleotide (i.e. a nucleotide which corresponds to the same position of a nucleotide of interest in the target nucleic acid) at the 3′ terminus of the primer. In such a case, the annealing of the 3′ terminal nucleotide of the primer may be dependent on the presence of the nucleotide of interest in the target nucleic acid. If the 3′ terminal nucleotide of the primer does not anneal with a nucleotide in the target nucleic acid (e.g. due to a mismatch between the nucleotides), the mismatch may significantly impair a nucleic acid polymerase from synthesizing an extension product from the primer. Accordingly, in some embodiments, a primer having a 3′ terminal nucleotide which corresponds to a nucleotide of interest may be useful for determining the presence or absence of a particular nucleotide in a target nucleic acid. In such embodiments, in some situations the critical nucleotide at the 3′ terminus of the primer may be selected to be complementary the nucleotide of interest in the target nucleic acid, and in some other situations the critical nucleotide at the 3′ terminus of the primer may be selected to be non-complementary the nucleotide of interest in the target nucleic acid. The nucleotide of interest may represent, for example, a wild-type form, a mutant form, or a polymorphism of a target nucleic acid.

In other embodiments, a particular nucleotide of interest in a target nucleic acid (e.g. a mutation or SNP) may be detected by selecting primers such that the nucleotide of interest is present in the target nucleic acid in a region which is not complementary to a template-binding region of a first or second primer. For example, the nucleotide of interest may be approximately in the middle of a target nucleic acid sequence. In embodiments, the nucleotide of interest may be in an “internal motif” described elsewhere herein. When a nucleotide of interest is in an internal motif, in embodiments, a primer pair may be prepared to contain a nucleotide sequence in the tail region of the primers which is complementary to an internal motif or the complement thereof, and which may be used to assay for the presence or absence of the nucleotide of interest in the internal motif in the target sequence. As explained elsewhere herein, the temporary annealing of a nucleotide sequence in the tail region of a primer to an internal motif in an extension product of that primer may increase the rate of a reaction provided herein. In some circumstances, the greater the affinity of a nucleotide sequence in the tail region of a primer to the internal motif in an extension product of that primer, the faster the reaction may occur. Also, typically, the greater the number of nucleotides in the nucleotide sequence in the tail region of the primer which can bind to nucleotides in the internal motif in the extension product of the primer, the greater the affinity of the nucleotide sequence in the tail region of the extension product for the internal motif in the extension product. Thus, with compositions and methods provided herein, in embodiments, the presence or absence of a nucleotide of interest in a target sequence may be determined through the use of primers which have a nucleotide sequence in the tail region of the primer which can bind to the internal motif in the target sequence or a complement thereof, and which, within the tail region, do or do not have a nucleotide which specifically binds with the particular nucleotide of interest in the internal motif or its complement. Typically, the reaction will occur faster when the nucleotide sequence in the tail region of a primer contains a nucleotide which is complementary to the nucleotide in the extension product of that primer which corresponds to the nucleotide of interest in the target, than when the relevant nucleotide in the tail region of the primer is not complementary to the nucleotide in the extension product of that primer which corresponds to the nucleotide of interest in the target. For example, the internal motif of a wild-type version of a target nucleic acid strand may have the nucleotide sequence: 5′ TATTGCAT 3′. However, the “G” in the sequence may frequently be mutated to an “A” in many individuals in the population. In order to determine whether a particular target nucleic acid contains a “G” or other nucleotide in the sequence, a primer may be prepared which contain a nucleotide sequence in its tail region having the sequence: 5′ATGCAATA 3′. If this primer (and an appropriate second primer) is used to amplify the target nucleic acid according to a method provided herein, the amplification reaction may have a certain reaction rate if the “G” is present in the internal motif, versus if a different nucleotide is present at the position of the “G” in the internal motif. This is because the nucleotide sequence in the tail region of the primer will be perfectly complementary to the internal motif if the “G” is present in the internal motif, but it will not be if a nucleotide other than a “G” is present at the G's normal position. Generally, in this example, if a “G” is present in the internal motif, the tail region of the primer will anneal to the internal motif more frequently than if the “G” is not present, and this may in turn lead to a faster reaction rate if the “G” is present than not present. With systems and methods provided herein, primers may be prepared to assay for either the presence or absence of a nucleotide of interest (e.g. primers can be prepared such that the reaction occurs more quickly if a mutated version of a nucleotide is present than if a wild-type version of the nucleotide is present, or vice-versa). Also, in embodiments, primers may be prepared so as to determine the identity of an unknown nucleotide in a position of interest in an internal motif in a target. For example, four different primer pairs may be prepared, each containing either an A, T, G, or C as appropriate corresponding to a nucleotide of interest in a target nucleic acid. In embodiments, the primer pair which yields the fastest reaction results may indicate which nucleotide is present in the position of interest in the internal motif in the target nucleic acid. For instance, a target nucleic acid may have an internal motif which commonly has the sequence, in the 5′ to 3′ direction of: GTAACGAG. However, different variants of the target nucleic acid may have different nucleotides in the 5th position in the internal motif (i.e. the position of the “C”). Continuing with the example, if a sample containing the target nucleic acid is provided, wherein it is unknown what nucleotide is in the 5^(th) position in the of the internal motif, the sample can be divided into at least 4 portions, and the four portions can be used for a first reaction mixture, a second reaction mixture, a third reaction mixture, and a fourth reaction mixture. To the first reaction mixture, a first primer pair is provided, where the primers have tail regions with the sequences, in the 5′ to 3′ direction: CTCGTTAC and GTAACGAG, respectively. These tail regions will be perfectly complementary to the internal motif or complement thereof, if a “C” is present at 5th position in internal motif. Thus, if the target is amplified most quickly in this reaction mixture, it indicates that a “C” is present in the position of the nucleotide of interest in the internal motif in the target. To the second reaction mixture, a second primer pair is provided, where the primers have tail regions with the sequences, in the 5′ to 3′ direction: CTCATTAC and GTAATGAG, respectively. These tail regions will be perfectly complementary to the internal motif or complement thereof, if a “T” is present at 5^(th) position in internal motif. Thus, if the target is amplified most quickly in this reaction mixture, it indicates that a “T” is present in the position of the nucleotide of interest in the internal motif in the target. To the third reaction mixture, a third primer pair is provided, where the primers have tail regions with the sequences, in the 5′ to 3′ direction: CTCCTTAC and GTAAGGAG, respectively. These tail regions will be perfectly complementary to the internal motif or complement thereof, if a “G” is present at 5th position in internal motif. Thus, if the target is amplified most quickly in this reaction mixture, it indicates that a “G” is present in the position of the nucleotide of interest in the internal motif in the target. To the fourth reaction mixture, a fourth primer pair is provided, where the primers have tail regions with the sequences, in the 5′ to 3′ direction: CTCTTTAC and GTAAAGAG, respectively. These tail regions will be perfectly complementary to the internal motif or complement thereof, if an “A” is present at 5th position in internal motif. Thus, if the target is amplified most quickly in this reaction mixture, it indicates that an “A” is present in the position of the nucleotide of interest in the internal motif in the target. In embodiments, methods or compositions as generally described above may optionally be prepared with only 2 or 3 reaction mixtures with primer pairs corresponding to only 2 or 3 options for a nucleotide at a position of interest, if for example, it is not desired to screen all possible nucleotide variants (for instance if only 2 different nucleotides are typically found in a position of interest).

In embodiments, a sample containing a target nucleic acid may be divided into at least a first and a second portion, where the first portion is incubated in a first reaction mixture with a first primer pair, and the second portion is incubated in a second reaction mixture with a second primer pair, according to methods provided herein. In embodiments, the first primer pair and the second primer pair differ from each other by only a single nucleotide in the respective tail/first regions of the primer (e.g. the sequence of the first region of the first primer of the first pair differs from the sequence of the first region of the first primer of the second pair by only a single nucleotide). The single nucleotide which is different in the tail region of the primers may correspond to the position of a nucleotide of interest in the target nucleic acid. By comparing the rate or quantity of amplification of the target nucleic acid in the first reaction mixture to that in the second reaction mixture, the identity of a nucleotide of interest in the target nucleic acid may be determined. In embodiments, the method outlined above may be performed with a first, second, third, and fourth portion of the sample, and with a first primer pair, second primer pair, third primer pair, and fourth primer pair, wherein the first primer pair, second primer pair, third primer pair, and fourth primer pair differ from each other by only a single nucleotide in the respective tail/first region of the primers, as described above.

In embodiments, at least a first target nucleic acid and a second target nucleic acid may be provided, wherein the first target nucleic acid and the second target nucleic acid differ by a single nucleotide at a position of interest in an internal motif as described elsewhere herein. In embodiments, the first target nucleic acid may be incubated in a first reaction mixture containing a primer pair described herein and the second target nucleic acid may be incubated in a second reaction mixture containing the same primer pair, according to methods provided herein. By comparing the rate or quantity of amplification of the first target nucleic acid in the first reaction mixture to that of the second target nucleic acid in second reaction mixture or to an absolute value, the identity of the nucleotide in the position of interest in one or both of the first target nucleic acid and the second target nucleic may be determined, according to principles described elsewhere herein.

In addition, in embodiments, compositions and methods described herein for detecting a single nucleotide of interest may also be used to detect multi-nucleotide mutations or polymorphisms.

Methods and compositions provided herein may be used to amplify a nucleic acid from any sample which may contain nucleic acids. Examples of samples may include various fluid samples. In some instances, the sample may be a bodily fluid sample from a subject. The sample may include one or more fluid component. In some instances, solid or semi-solid samples may be provided. The sample may include tissue collected from the subject. The sample may include a bodily fluid, secretion, or tissue of a subject. The sample may be a biological sample. The biological sample may be a bodily fluid, a secretion, or a tissue sample. Examples of biological samples may include but are not limited to, blood, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk or other excretions. The sample may be provided from a human or animal. Samples may be from a plant, microorganism (e.g. virus, bacteria), or other biological material.

In some embodiments, methods and compositions provided herein may be performed at or used at point of service locations (e.g. a subject's home or work, grocery stores, drug stores, clinics, schools, etc.). Methods and compositions provided herein may permit the rapid amplification of nucleic acids in a sample from a subject, in order to aid in the diagnosis or treatment of a subject. For example, methods and compositions provided here may be used test a sample from a subject for the presence of nucleic acid from a pathogen, such as a virus (e.g. influenza) or bacteria (e.g. streptococcus).

In embodiments, methods and compositions provided herein may be used to detect a pathogen, or a feature of a pathogen (e.g. a gene or gene variant within the pathogen). Pathogens or features thereof which may be detected with a method or composition provided herein may include, for example, Acinetobacter baumannii, blaROB resistance gene, blaTEM resistance gene, Bordetalla holmesii, Burkholderia cepacia, Candida albicans, Clostridium difficile, Clostridium sordellii, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecaelis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, KPC resistance gene, Legionella pneumophila, mecA resistance gene, mecC resistance gene, Moraxella catarrhalis, Mycobacterium abscessus, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus aureus (Vancomycin-resistant) VRSA, Streptococcus agalactiae (Strep B), Streptococcus pneumonia, Streptococcus pneumonia (penicillin-sensitive), Streptococcus pyogenes, vanA resistance gene, vanB resistance gene, Adenovirus B, Adenovirus C, Adenovirus E, Bocavirus 1+3, Bocavirus 2+4, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumonia, Clostridium sordellii, Coronavirus 229E, Coronavirus HKU1, Coronavirus MERS, Coronavirus NL63, Coronavirus OC43, Human metapneumovirus A, Human metapneumovirus B, Influenza A-MP, Influenza B-MP, Influenza H1N1 (2009), Influenza H1N1 (seasonal), Influenza H3N2, Influenza H5N1, Influenza H7N9 (HA gene), Influenza H7N9 (NA gene), measles virus, mumps virus, Mycobacterium tuberculosis, Mycoplasma pneumonia, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4a, Parainfluenza 4b, Respiratory syncytial virus A (RSV A), Respiratory syncytial virus B (RSV B), Rhinovirus A, Rhinovirus B, Rhinovirus C, rubella virus, Streptococcus pyogenes, Candida albicans, Chlamydia trachomatis, human papilloma virus, HPV 16, HPV 18, HSV-1/2, Mycoplasma genitalium, Neisseria gonorrhoeae, Treponema pallidum (syphilis), Trichomonas vaginalis, Varicella-zoster virus, Epstein-Barr Virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, HIV, HIV-1 group M HIV-1 group 01, HIV-1 group 03, HIV-2 group A, HIV-2 group B, Mycoplasma hominis, Proteus mirabilis, Proteus penneri, Proteus vulgaris, S. saprophyticus, Ureaplasma urealyticum, Bacillus anthracis, Yersina pestis, Dengue virus, Plasmodium, Tryponosoma cruzi (chagas), West Nile Virus 1, West Nile Virus 2, Norovirus lineage 1, Norovirus lineage 2, Ebola virus. In embodiments, 1, 2, 3, 5, 10, 20, 30, 50, or more of such pathogens or features thereof may be assayed for from a single sample from a subject using a method provided herein. In embodiments, any of the nucleic acid amplification-based assays or assay panels as described in International Application No. PCT/US2014/54424, filed Sep. 5, 2014 or U.S. Provisional Patent Application No. 62/001,050, filed May 20, 2014, which are hereby incorporated by reference in its entirety for all purposes, may be performed using a method for nucleic acid amplification described herein. Similarly, any of the pathogens or features thereof described in International Application No. PCT/US2014/54424 or U.S. Provisional Patent Application No. 62/001,050 may be assayed for with a nucleic acid amplification method provided herein.

In embodiments, an influenza panel may be performed using methods provided herein for amplification of nucleic acids from one or more strains of influenza. For example, using methods provided herein, one or more nucleic acids from one or more of Influenza A, Influenza A H3N2, Influenza A H5N1, Influenza A H7N9-HA gene, Influenza A H7N9-NA gene, or Influenza B may be amplified. Other panels that may be performed using methods provided herein include, for example, a respiratory panel, a sexually transmitted disease panel, a blood-borne pathogen panel, or a hospital acquired infections panel. Any of these panels may include assays for at least 2, 3, 4, 5, 10, 15, or 20 different pathogens or genes.

In embodiments, methods and compositions provided herein may be used for Influenza A virus detection and sub-typing. Influenza A subtypes are defined by the type of two viral genes, hemagglutin (HA) and neuraminidase (NA). Presently, there are 18 known HA genes (H1-H18) and 11 known NA genes (N1-N11). These genes are encoded on different molecules in the Influenza A virus, and thus it is possible for an Influenza A subtype to carry any combination of different HA and NA gene. In order to accurately detect and type an Influenza A virus, it may be desirable to test a sample which may or is known to contain Influenza A virus for at least two and up to all of the 18 known HA genes and at least two and up to all the 11 known NA genes. In embodiments, a single sample from a subject may be divided into two or more portions, and the portions may be incubated under reaction conditions provided herein with primer pairs having template-binding regions specific for each of the 18 known HA genes and 11 known NA genes (i.e. 29 different primer pairs). In embodiments, a single sample from a subject may be divided into at least 29 different portions, wherein the first portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H1, wherein the second portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H2, wherein the third portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H3, wherein the fourth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H4, wherein the fifth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H5, wherein the sixth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H6, wherein the seventh portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H7, wherein the eighth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H8, wherein the ninth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H9, wherein the tenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H10, wherein the eleventh portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H11, wherein the twelfth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H12, wherein the thirteen portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H13, wherein the fourteenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H14, wherein the fifteenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H15, wherein the sixteenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H16, wherein the seventeenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H17, wherein the eighteenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for H18, wherein the nineteenth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N1, wherein the twentieth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N2, wherein the twenty-first portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N3, wherein the twenty-second portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N4, wherein the twenty-third portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N5, wherein the twenty-fourth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N6, wherein the twenty-fifth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N7, wherein the twenty-sixth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N8, wherein the twenty-seventh portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N9, wherein the twenty-eighth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N10, and wherein the twenty-ninth portion is incubated under reaction conditions provided herein with a primer pair having template-binding regions specific for N11. In some embodiments, primer pairs for 2, 3, 4, 5, or more different HA or NA subtypes may be incubated with a single portion of a sample from a subject (e.g. in a common vessel), and the amplification reaction products may be identified using a sequence-specific probe (e.g. DNA probe or PNA probe), as described elsewhere herein. In embodiments, a panel may be performed wherein a single sample is used for amplification reactions with primer pairs for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or all 29 of the HA and NA subtypes described above. In embodiments, a panel may include reactions to amplify one or more of H1, H2, H3, H5, H7, or N9. In embodiments, a panel or method described herein for the detection of Influenza A or subtypes thereof may further include reagents or reactions to detect, for example, Influenza A matrix protein gene (this is present in all Influenza A subtypes, and thus may be used to identify Influenza A in a sample), human RNaseP (to ensure adequate sample collection from a human subject; for non-human subjects, other appropriate species-specific control genes may be used), or a spike-in nucleic acid control, which may be added at one or more points during a sample collection or processing step, in order to ensure adequate sample collection or processing. In embodiments, kits may be provided to perform any of the methods or for any of the panels provided above.

In embodiments, using methods provided herein, a single pair of primers may be used to amplify a greater variety of target nucleic acid sequences than may be amplified with a single pair of primers using other nucleic acid amplification methods (e.g. PCR or LAMP). For example, in embodiments, the template-binding regions of primers provided herein may sufficiently anneal to templates to amplify the templates according to methods provided herein even when there are 1, 2, 3, 4, 5 or more nucleotide mismatches between the template-binding region of the primer and the template. In embodiments, methods provided herein may be performed when at least 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24% of the nucleotides in the template binding region of at least a first primer and optionally also the second primer used in a method provided are not complementary to corresponding nucleotides in the template sequence. Use of primers and methods provided herein, therefore, may facilitate the amplification of multiple similar but different sequences by the same pair of primers. For instance, primers and methods provided herein may be useful for the detection of multiple different strains of influenza A, where the strains have similar but different nucleotide sequences. For example, there may be two different strains of Influenza A, where both strains are defined as H1N1 sub-type, but the strains have slightly different H1 or N1 nucleotide sequences. Using compositions and methods provided herein, in embodiments, both the first strain and the second strain may be amplified using the same primer pair.

In addition, primers used for the amplification of nucleic acids from pathogens may be selected based on highly conserved regions in nucleic acids of pathogens, to further aid in the detection multiple different strains of a pathogen with the same primer pair according to methods provided herein. Such highly conserved regions may be identified, for example, by sequence alignment of a gene of interest across multiple strains of a pathogen. For instance, HA3 sequences with Genbank ID numbers: AEK98400, ABG47862, AE000689, ABF17954, AFG99237, AFG99027, AB033069, ABE12623, AFG72790, AAA87553, AAZ38605, AFN19700, ABB04371, ABC39805 are from different isolates of H3N2 Influenza A virus. These HA3 genes each have slightly different nucleotide sequences. Nonetheless, through careful selection of primers, all such HA3 genes may be amplified with the same set of primers according to a method provided herein. For example, a portion of the HA3 gene having the sequence, in 5′-3′ order:

(SEQ ID NO: 91) AACCTCACTACGTAAGTCTTGACGTAAACGGTTACCCACGTAGACTAGAG TAATAACTCGAAAAGGGT may be selected as a target for amplification according to a method provided herein (the sequence is provided in DNA, rather than RNA form). Describing this sequence, in the 5′ to 3′ direction, the first underlined sequence the second portion of the template, the second underlined sequence is the internal motif, and the third underlined sequence is the first portion of the template, as these terms are used, for example in FIG. 10. In this and other such examples, both the total target sequence for amplification, as well as the particular sequences to function as the first portion of the template, second portion of the template, and internal motif may be selected such that primers as described herein may be used to successfully amplify a wide range of related sequences according to methods provided herein. In embodiments, a first primer as described herein may be provided wherein the template binding region of the primer is complementary to the first portion of the above template, and a second primer as described herein may be provided wherein the template binding region of the primer is complementary to a partner sequence which is complementary to the second portion of the template. Optionally, the primers may further have tail regions which are complementary to the internal motif or a complementary sequence thereof. Such primers may be provided, for example, in compositions or methods provided herein.

With methods provided herein pathogens or genes may be positively identified at concentrations as low as, for example, less than 1000, 500, 100, 50, 10, 5, 2, or 1 copy per microliter in a sample or a reaction mixture. In embodiments, methods provided herein may specifically amplify nucleic acid from one type of pathogen or gene and not amplify nucleic acid from a related pathogen or gene. For example, assays provided herein to amplify Influenza A matrix protein gene may amplify this gene from multiple different strains of Influenza A, but not amplify genetic material from Influenza B.

In embodiments, methods provided herein may be successfully performed in the presence of one or more potentially interfering substances. Examples of potentially interfering substances include BSA, glucose, bilirubin, nitrites, beta-hCG, acetone, low pH conditions, high pH conditions, acetaminophen, aspirin, progestin, ethinyl estradiol, urine preservatives, seminal fluid, personal lubricants, contraceptive jellies, spermicides, feminine powders, hemorrhoid creams, miconzole, human genomic DNA, lotions, universal transport media (viral), amies transport media (bacteria), blood, mucin, acyclovir, cold sore treatments, urine, feces, hemoglobin, triglycerides, EDTA, heparin, cholesterol, gamma-globulin, ampicillin, nicotine, cotinine, nasal sprays, nasal drops, or any combination thereof. In embodiments, methods provided herein may be performed in the presence of a potentially interfering substance which is at a concentration of up to at least as great as provided in U.S. Provisional Patent Application No. 62/001,050, filed May 20, 2014, which is hereby incorporated by reference for all purposes. In embodiments, methods provided herein may be performed in the presence of a potentially interfering substance which is at a concentration of up to at least 10%, 25%, 50%, or 100% greater than provided in U.S. Provisional Patent Application No. U.S. Provisional Patent Application No. 62/001,050.

The assays and methods disclosed herein may be performed on a device, or on a system, for processing a sample. The assays and methods disclosed herein can be readily incorporated into and used in a device for processing a sample, or a system for processing a sample, which may be an automated assay device, or may be an automated assay system. Such a device, and such a system, may be useful for the practice of the methods disclosed herein. For example, a device may be useful for receiving a sample. A device may be useful for preparing, or for processing a sample. A device may be useful for performing an assay on a sample. A device may be useful for obtaining data from a sample. A device may be useful for transmitting data obtained from a sample. A device may be useful for disposing of a sample following processing or assaying of a sample.

A device may be part of a system, a component of which may be a sample processing device. A device may be a sample processing device. A sample processing device may be configured to facilitate collection of a sample, prepare a sample for a clinical test, or perform a method with one or more reagents, as disclosed herein. A sample processing device may be configured to obtain data from a sample. A sample processing device may be configured to transmit data obtained from a sample. A sample processing device may be configured to analyze data from a sample. A sample processing device may be configured to communicate with another device, or a laboratory, or an individual affiliated with a laboratory, to analyze data obtained from a sample.

A sample processing device may be configured to be placed in or on a subject. A sample processing device may be configured to accept a sample from a subject, either directly or indirectly. A sample may be, for example, a blood sample (e.g., a sample obtained from a fingerstick, or from venipuncture, or an arterial blood sample), a urine sample, a biopsy sample, a tissue slice, stool sample, or other biological sample; a water sample, a soil sample, a food sample, an air sample; or other sample. A blood sample may comprise, e.g., whole blood, plasma, or serum. A sample processing device may receive a sample from the subject through a housing of the device. The sample collection may occur at a sample collection site, or elsewhere. The sample may be provided to the device at a sample collection site.

In some embodiments, a sample processing device may be configured to accept or hold a cartridge. In some embodiments, a sample processing device may comprise a cartridge. The cartridge may be removable from the sample processing device. In some embodiments, a sample may be provided to the cartridge of the sample processing device. Alternatively, a sample may be provided to another portion of a sample processing device. The cartridge and/or device may comprise a sample collection unit that may be configured to accept a sample.

A cartridge may include a sample, and may include reagents for use in processing or testing a sample, disposables for use in processing or testing a sample, or other materials. A cartridge may contain reagents disclosed herein for the performing a method disclosed herein. Following placement of a cartridge on, or insertion of a cartridge into, a sample processing device, one or more components of the cartridge may be brought into fluid communication with other components of the sample processing device. For example, if a sample is collected at a cartridge, the sample may be transferred to other portions of the sample processing device. Similarly, if one or more reagents are provided on a cartridge, the reagents may be transferred to other portions of the sample processing device, or other components of the sample processing device may be brought to the reagents. In some embodiments, the reagents or components of a cartridge may remain on-board the cartridge. In some embodiments, no fluidics are included that require tubing or that require maintenance (e.g., manual or automated maintenance).

A sample or reagent may be transferred to a device, such as a sample processing device. A sample or reagent may be transferred within a device. Such transfer of sample or reagent may be accomplished without providing a continuous fluid pathway from cartridge to device. Such transfer of sample or reagent may be accomplished without providing a continuous fluid pathway within a device. In embodiments, such transfer of sample or reagent may be accomplished by a sample handling system (e.g., a pipette); for example, a sample, reagent, or aliquot thereof may be aspirated into an open-tipped transfer component, such as a pipette tip, which may be operably connected to a sample handling system which transfers the tip, with the sample, reagent, or aliquot thereof contained within the tip, to a location on or within the sample processing device. The sample, reagent, or aliquot thereof can be deposited at a location on or within the sample processing device. Sample and reagent, or multiple reagents, may be mixed using a sample handling system in a similar manner. One or more components of the cartridge may be transferred in an automated fashion to other portions of the sample processing device, and vice versa.

A device, such as a sample processing device, may have a fluid handling system. A fluid handling system may perform, or may aid in performing, transport, dilution, extraction, aliquotting, mixing, and other actions with a fluid, such as a sample. In some embodiments, a fluid handling system may be contained within a device housing. A fluid handling system may permit the collection, delivery, processing and/or transport of a fluid, dissolution of dry reagents, mixing of liquid and/or dry reagents with a liquid, as well as collection, delivery, processing and/or transport of non-fluidic components, samples, or materials. The fluid may be a sample, a reagent, diluent, wash, dye, or any other fluid that may be used by the device, and may include, but not limited to, homogenous fluids, different liquids, emulsions, suspensions, and other fluids. A fluid handling system, including without limitation a pipette, may also be used to transport vessels (with or without fluid contained therein) around the device. The fluid handling system may dispense or aspirate a fluid. The sample may include one or more particulate or solid matter floating within a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipette tip, syringe, capillary, or other component. The fluid handling system may have a portion with an interior surface and an exterior surface and an open end. The fluid handling system may comprise a pipette, which may include a pipette body and a pipette nozzle, and may comprise a pipette tip. A pipette tip may or may not be removable from a pipette nozzle. In embodiments, a fluid handling system may use a pipette mated with a pipette tip; a pipette tip may be disposable. A tip may form a fluid-tight seal when mated with a pipette. A pipette tip may be used once, twice, or more times. In embodiments, a fluid handling system may use a pipette or similar device, with or without a pipette tip, to aspirate, dispense, mix, transport, or otherwise handle the fluid. The fluid may be dispensed from the fluid handling system when desired. The fluid may be contained within a pipette tip prior to being dispensed, e.g., from an orifice in the pipette tip. In embodiments, or instances during use, all of the fluid may be dispensed; in other embodiments, or instances during use, a portion of the fluid within a tip may be dispensed. A pipette may selectively aspirate a fluid. The pipette may aspirate a selected amount of fluid. The pipette may be capable of actuating stirring mechanisms to mix the fluid within the tip or within a vessel. The pipette may incorporate tips or vessels creating continuous flow loops for mixing, including of materials or reagents that are in non-liquid form. A pipette tip may also facilitate mixture by metered delivery of multiple fluids simultaneously or in sequence, such as in 2-part substrate reactions.

The fluid handling system may include one or more fluidically isolated or hydraulically independent units. For example, the fluid handling system may include one, two, or more pipette tips. The pipette tips may be configured to accept and confine a fluid. The tips may be fluidically isolated from or hydraulically independent of one another. The fluid contained within each tip may be fluidically isolated or hydraulically independent from fluids in other tips and from other fluids within the device. The fluidically isolated or hydraulically independent units may be movable relative to other portions of the device and/or one another. The fluidically isolated or hydraulically independent units may be individually movable. A fluid handling system may comprise one or more base or support. A base or support may support one or more pipette or pipette units. A base or support may connect one or more pipettes of the fluid handling system to one another.

A sample processing device may be configured to perform processing steps or actions on a sample obtained from a subject. Sample processing may include sample preparation, including, e.g., sample dilution, division of a sample into aliquots, extraction, contact with a reagent, filtration, separation, centrifugation, or other preparatory or processing action or step. A sample processing device may be configured to perform one or more sample preparation action or step on the sample. Optionally, a sample may be prepared for a chemical reaction and/or physical processing step. A sample preparation action or step may include one or more of the following: centrifugation, separation, filtration, dilution, enriching, purification, precipitation, incubation, pipetting, transport, chromatography, cell lysis, cytometry, pulverization, grinding, activation, ultrasonication, micro column processing, processing with magnetic beads, processing with nanoparticles, or other sample preparation action or steps. For example, sample preparation may include one or more step to separate blood into serum and/or particulate fractions, or to separate any other sample into various components. Sample preparation may include one or more step to dilute and/or concentrate a sample, such as a blood sample, or other biological samples. Sample preparation may include adding an anti-coagulant or other ingredients to a sample. Sample preparation may also include purification of a sample. In embodiments, all sample processing, preparation, or assay actions or steps are performed by a single device. In embodiments, all sample processing, preparation, or assay actions or steps are performed within a housing of a single device. In embodiments, most sample processing, preparation, or assay actions or steps are performed by a single device, and may be performed within a housing of a single device. In embodiments, many sample processing, preparation, or assay actions or steps are performed by a single device, and may be performed within a housing of a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.

A sample processing device may be configured to run one or more assays on a sample, and to obtain data from the sample. A sample processing device may perform methods provided herein, as well as additional assays. An assay may include one or more physical or chemical treatments, and may include running one or more chemical or physical reactions. A sample processing device may be configured to perform one, two or more assays on a small sample of bodily fluid. One or more chemical reaction may take place on a sample having a volume, as described elsewhere herein. For example, one or more chemical reaction may take place in a pill having less than femtoliter volumes. In an instance, the sample collection unit is configured to receive a volume of the bodily fluid sample equivalent to a single drop or less of blood or interstitial fluid. In embodiments, the volume of a sample may be a small volume, where a small volume may be a volume that is less than about 1000 μL, or less than about 500 μL, or less than about 250 μL, or less than about 150 μL, or less than about 100 μL, or less than about 75 μL, or less than about 50 μL, or less than about 40 μL, or less than about 20 μL, or less than about 10 μL, less than about 5 μL, less than about 1 μL, less than about 0.5 μL, less than about 0.1 μL, or other small volume. In embodiments, all sample assay actions or steps are performed on a single sample. In embodiments, all sample assay actions or steps are performed by a single device. In embodiments, all sample assay actions or steps are performed within a housing of a single device. In embodiments, most sample assay actions or steps are performed by a single device, and may be performed within a housing of a single device. In embodiments, many sample assay actions or steps are performed by a single device, and may be performed within a housing of a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.

A sample processing device may be configured to perform a plurality of assays on a sample. In some embodiments, a sample processing device may be configured to perform a method provided herein and one, two, or more additional assays. In embodiments, a sample processing device may be configured to perform a plurality of assays on a single sample. In embodiments, a sample processing device may be configured to perform a plurality of assays on a single sample, where the sample is a small sample. For example, a small sample may have a sample volume that is a small volume of less than about 1000 μL, or less than about 500 μL, or less than about 250 μL, or less than about 150 μL, or less than about 100 μL, or less than about 75 μL, or less than about 50 μL, or less than about 40 μL, or less than about 20 μL, or less than about 10 μL, less than about 5 μL, less than about 1 μL, less than about 0.5 μL, less than about 0.1 μL, or other small volume. A sample processing device may be capable of performing multiplexed assays on a single sample. A plurality of assays may be run simultaneously; may be run sequentially; or some assays may be run simultaneously while others are run sequentially. One or more control assays and/or calibrators (e.g., including a configuration with a control of a calibrator for the assay/tests) can also be incorporated into the device; control assays and assay on calibrators may be performed simultaneously with assays performed on a sample, or may be performed before or after assays performed on a sample, or any combination thereof. In embodiments, all sample assay actions or steps are performed by a single device. In embodiments, all of a plurality of assay actions or steps are performed within a housing of a single device. In embodiments, most sample assay actions or steps, of a plurality of assays, are performed by a single device, and may be performed within a housing of a single device. In embodiments, many sample assay actions or steps, of a plurality of assays, are performed by a single device, and may be performed within a housing of a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.

In embodiments, all of a plurality of assays may be performed in a short time period. In embodiments, such a short time period comprises less than about three hours, or less than about two hours, or less than about one hour, or less than about 40 minutes, or less than about 30 minutes, or less than about 25 minutes, or less than about 20 minutes, or less than about 15 minutes, or less than about 10 minutes, or less than about 5 minutes, or less than about 4 minutes, or less than about 3 minutes, or less than about 2 minutes, or less than about 1 minute, or other short time period.

A sample processing device may be configured to detect one or more signals relating to the sample. A sample processing device may be configured to identify one or more properties of the sample. For instance, the sample processing device may be configured to detect the presence or concentration of one analyte (e.g. a target nucleic acid) or a plurality of analytes or a disease condition in the sample (e.g., in or through a bodily fluid, secretion, tissue, or other sample). Alternatively, the sample processing device may be configured to detect a signal or signals that may be analyzed to detect the presence or concentration of one or more analytes (which may be indicative of a disease condition) or a disease condition in the sample. The signals may be analyzed on board the device, or at another location. Running a clinical test may or may not include any analysis or comparison of data collected.

A chemical reaction or other processing steps may be performed, with or without the sample. Examples of steps, tests, or assays that may be prepared or run by the device may include, but are not limited to immunoassay, nucleic acid assay (e.g. methods provided herein), receptor-based assay, cytometric assay, colorimetric assay, enzymatic assay, electrophoretic assay, electrochemical assay, spectroscopic assay, chromatographic assay, microscopic assay, topographic assay, calorimetric assay, turbidimetric assay, agglutination assay, radioisotope assay, viscometric assay, coagulation assay, clotting time assay, protein synthesis assay, histological assay, culture assay, osmolarity assay, and/or other types of assays, centrifugation, separation, filtration, dilution, enriching, purification, precipitation, pulverization, incubation, pipetting, transport, cell lysis, or other sample preparation action or steps, or combinations thereof. Steps, tests, or assays that may be prepared or run by the device may include imaging, including microscopy, cytometry, and other techniques preparing or utilizing images. Steps, tests, or assays that may be prepared or run by the device may further include an assessment of histology, morphology, kinematics, dynamics, and/or state of a sample, which may include such assessment for cells.

A device may be capable of performing all on-board steps (e.g., steps or actions performed by a single device) in a short amount of time. A device may be capable of performing all on-board steps on a single sample in a short amount of time. For example, from sample collection from a subject to transmitting data and/or to analysis may take about 3 hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less. The amount of time from accepting a sample within the device to transmitting data and/or to analysis from the device regarding such a sample may depend on the type or number of steps, tests, or assays performed on the sample. The amount of time from accepting a sample within the device to transmitting data and/or to analysis from the device regarding such a sample may take about 3 hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less.

A device may be configured to prepare a sample for disposal, or to dispose of a sample, such as a biological sample, following processing or assaying of a sample.

In embodiments, a sample processing device may be configured to transmit data obtained from a sample. In embodiments, a sample processing device may be configured to communicate over a network. A sample processing device may include a communication module that may interface with the network. A sample processing device may be connected to the network via a wired connection or wirelessly. The network may be a local area network (LAN) or a wide area network (WAN) such as the Internet. In some embodiments, the network may be a personal area network. The network may include the cloud. The sample processing device may be connected to the network without requiring an intermediary device, or an intermediary device may be required to connect a sample processing device to a network. A sample processing device may communicate over a network with another device, which may be any type of networked device, including but not limited to a personal computer, server computer, or laptop computer; personal digital assistants (PDAs) such as a Windows CE device; phones such as cellular phones, smartphones (e.g., iPhone, Android, Blackberry, etc.), or location-aware portable phones (such as GPS); a roaming device, such as a network-connected roaming device; a wireless device such as a wireless email device or other device capable of communicating wireless with a computer network; or any other type of network device that may communicate possibly over a network and handle electronic transactions. Such communication may include providing data to a cloud computing infrastructure or any other type of data storage infrastructure which may be accessed by other devices.

A sample processing device may provide data regarding a sample to, e.g., a health care professional, a health care professional location, such as a laboratory, or an affiliate thereof. One or more of a laboratory, health care professional, or subject may have a network device able to receive or access data provided by the sample processing device. A sample processing device may be configured to provide data regarding a sample to a database. A sample processing device may be configured to provide data regarding a sample to an electronic medical records system, to a laboratory information system, to a laboratory automation system, or other system or software. A sample processing device may provide data in the form of a report.

A laboratory, device, or other entity or software may perform analysis on data regarding a sample in real-time. A software system may perform chemical analysis and/or pathological analysis, or these could be distributed amongst combinations of lab, clinical, and specialty or expert personnel. Analysis may include qualitative and/or quantitative evaluation of a sample. Data analysis may include a subsequent qualitative and/or quantitative evaluation of a sample. Optionally, a report may be generated based on raw data, pre-processed data, or analyzed data. Such a report may be prepared so as to maintain confidentiality of the data obtained from the sample, the identity and other information regarding the subject from whom a sample was obtained, analysis of the data, and other confidential information. The report and/or the data may be transmitted to a health care professional. Data obtained by a sample processing device, or analysis of such data, or reports, may be provided to a database, an electronic medical records system, to a laboratory information system, to a laboratory automation system, or other system or software.

Description and disclosure of examples of reagents, assays, methods, kits, devices, and systems which may use, or be used with, methods, compositions, or other reagents disclosed herein may be found, for example, in U.S. Pat. Nos. 8,088,593; 8,380,541; U.S. patent application Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. patent application Ser. No. 13/244,947 filed Sep. 26, 2011; International Application No. PCT/US2012/57155, filed Sep. 25, 2012; U.S. application Ser. No. 13/244,946, filed Sep. 26, 2011; U.S. patent application Ser. No. 13/244,949, filed Sep. 26, 2011; and U.S. Application Ser. No. 61/673,245, filed Sep. 26, 2011, International Application No. PCT/US14/30034, filed Mar. 15, 2014, and U.S. patent application Ser. No. 14/214,850, filed Mar. 15, 2014, the disclosures of which patents and patent applications are all hereby incorporated by reference in their entireties.

This application claims the benefit of, and priority to U.S. Provisional Patent Application No. 61/800,606, filed Mar. 15, 2013, U.S. Provisional Patent Application No. 61/908,027, filed Nov. 22, 2013; U.S. Provisional Patent Application No. 62/001,050, filed May 20, 2014; U.S. Non-Provisional patent application Ser. No. 14/214,850, filed Mar. 15, 2014; International Patent Application No. PCT/US14/30034, filed Mar. 15, 2014; International Patent Application No. PCT/US14/56151, filed Sep. 17, 2014; U.S. Non-Provisional patent application Ser. No. 14/546,998, filed Nov. 18, 2014; and U.S. Non-Provisional patent application Ser. No. 14/850,608, filed Sep. 10, 2015, the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.

EXAMPLES

The following examples are offered for illustrative purposes only, and are not intended to limit the present disclosure in any way.

Example 1—Amplification of a Template Nucleic Acid with Primers of Variable Tail Region Length

A method as provided herein was used to amplify a target nucleic acid. Reactions were prepared to assay for about an 102 nucleotide portion of target nucleic acid T124A1, which is a 464 nucleotide portion of an influenza A virus hemagglutinin (HA3) gene (an RNA molecule). The nucleotide sequence of T124A1 is provided in SEQ ID NO: 89. 12 variants of a first primer (“P1”) and 12 variants of a second primer (“P2”) were prepared. The sequences of all of the primer variants are provided in FIG. 2A. All of the primers contained a template-binding region 15 nucleotides in length. The sequence of the template-binding region of all of the variants of the first primer was: CAAACCGTACCAACC (SEQ ID NO: 80) (in the 5′-3′ direction). The sequence of the template-binding region of all of the variants of the second primer was: ATGCGGAATGTACC (SEQ ID NO: 81) (in the 5′-3′ direction). The different primer variants contained tail regions ranging from 8 to 22 nucleotides in length, specifically 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or 22 nucleotides in length. The tail region of the first primer had a base sequence of: CGCCGGATGGCTCTTGGGAAAC (SEQ ID NO: 82) (in the 5′-3′ direction). The base sequence is 22 nucleotides in length; the shorter tail regions of the first primer have the same sequence, minus the appropriate number of nucleotides from the 5′ end of the tail region (e.g. the primer with the 18 nucleotide length tail region has a tail region with the same sequence as the base sequence, minus the first 4 nucleotides (CGCC) of the base sequence). The tail region of the second primer had a base sequence of: GTTTCCCAAGAGCCATCCGGCG (SEQ ID NO: 83) (in the 5′-3′ direction). The base sequence is 22 nucleotides in length; the shorter tail regions of the first primer have the same sequence, minus the appropriate number of nucleotides from the 3′ end of the tail region (e.g. the primer with the 18 nucleotide length tail region has a tail region with the same sequence as the base sequence, minus the last 4 nucleotides (GGCG) of the base sequence).

150 microliter reaction mixtures were prepared, each containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 30 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 2 uM SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/μl murine RNase inhibitor (New England Biolabs), 0.8 μM of a first primer variant, 0.8 μM of a second primer variant, and 100,000 copies T124A1 template per microliter, and incubated at 59 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). The inflection points for the assays were determined using a single-threshold method with CFX Manager software (Bio-Rad), and are shown in FIG. 2B. The X-axis provides the length in nucleotides of the tail region of the first and second primer used in the reaction, and the Y-axis provides the inflection time (in minutes) of the assay. For each tail region nucleotide length, two adjacent bars are shown: the left bar is the inflection time for the reaction containing template, and the right bar is the inflection time for the reaction lacking template [“no template control” (“NTC”)]. For inflection times over 90 minutes, no bar is shown. As shown in FIG. 2B, under these reaction conditions, primers with tail regions of 8-15 nucleotides, and, more particularly, 8-11 nucleotides supported the fastest inflection times. The no template control reactions eventually show an inflection time; this is due to background non-specific products that are formed over time.

Example 2—Amplification of a Template Nucleic Acid with Primers of Variable Tail Region Length and Variable Template-Binding Region Length

A method as provided herein was used to amplify a target nucleic acid. Reactions were prepared to assay for target nucleic acid T124A1, which is described above in Example 1. Two different groups of first primer and second primer pair sets were prepared. In the first group of primer pairs, each of the primers had a template binding region 20 nucleotides in length (the “20 nucleotide template binding region” group). In the second group of primer pairs, each of the primers had a template binding region 16 nucleotides in length (the “16 nucleotide template binding region” group). Within each group, first and second primer pair sets with 8 different tail region lengths were prepared: 7, 8, 9, 10, 11, 12, 14, and 16 nucleotides. FIG. 3 provides the nucleotide sequences of the different primer pair sets for the 16 nucleotide template binding region group (FIG. 3A) and the 20 nucleotide template binding region group (FIG. 3B). In FIGS. 3A and 3B, the “tail” sequence refers to the tail region, and the “primer” region refers to the template-binding region of the primer. The sequences of the primers are shown in the 5′-3′ direction.

110 microliter reaction mixtures were prepared, each containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 30 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 2 uM SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/μl murine RNase inhibitor (New England Biolabs), 0.8 μM of a first primer variant, 0.8 μM of a second primer variant, and 100,000 copies T124A1 template per microliter, and incubated at 56 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). The inflection points for the assays are shown in FIG. 3C (20 nucleotide template binding region group) and 3D (16 nucleotide template binding region group). In both FIGS. 3C and 3D, the X-axis provides the length in nucleotides of the tail region of the first and second primer used in the reaction, and the Y-axis provides the inflection time (in minutes) of the assay. For each tail region nucleotide length, two adjacent bars are shown: the left bar is the inflection time for the reaction containing template, and the right bar is the inflection time for the reaction lacking template. As shown in FIG. 3, under these reaction conditions, reactions containing primers having relatively shorter tail regions showed faster inflection times and greater separation between the inflection time of reactions containing template versus reactions without template. For example, for both the 16 bp and 20 bp primers, reactions containing primers having a tail region length of 7, 8, or 9 nucleotides showed faster inflection times than reactions containing primers having a tail region length of 12, 14, or 16 nucleotides.

Example 3—Amplification of a Template Nucleic Acid at Variable Temperatures

A method as provided herein was used to amplify a target nucleic acid. Reactions were prepared to assay for target nucleic acid T124A1, described above in Example 1. First primer “RLX0892” (sequence: 5′ TTGGGAAACCAAACCGTACCAACC 3′)(SEQ ID NO: 20) and second primer “RLX0893” (sequence: 5′ GTTTCCCAAATGCGGAATGTACC 3′)(SEQ ID NO: 32) were used to amplify T124A1. Both of these primers have a 9 nucleotide tail region, RLX0892 has a 15 nucleotide template-binding region and RLX0893 has a 14 nucleotide template-binding region. The tail region of RLX0892 is: 5′ TTGGGAAAC 3′ (SEQ ID NO: 84) and the tail region of RLX0893 is: 5′ GTTTCCCAA 3′ (SEQ ID NO: 85).

80 microliter reaction mixtures were prepared, each containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 20 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 2 uM SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/μl murine RNase inhibitor (New England Biolabs), 0.8 μM of first primer RLX0892, 0.8 μM of second primer RLX0893, and 10,000, 1,000, 100 or 0 copies T124A1 template per microliter, and incubated in triplicate at 52, 52.7, 54, 55.9, 58.4, 60.3, 61.4 or 62 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). The inflection points for the assays are shown in FIG. 4. The X-axis provides the incubation temperature of the reaction, and the Y-axis provides the inflection time (in minutes) of the assay. For each temperature, 4 adjacent bars are shown, from left to right: 10,000 copies template/microliter, 1000 copies template/microliter, 100 copies template/microliter, or no template control. As shown in FIG. 4, under these reaction conditions, the assays effectively amplified the template at 1000 copies template/microliter across the full range of temperatures from 52-62 C, and at some temperatures, at concentrations of template at least as low as 100 copies template/microliter.

Example 4—Amplification of a Template Nucleic Acid in the Presence of Human Genomic DNA

A method as provided herein was used to amplify a target nucleic acid. Reactions were prepared to assay for target nucleic acid T124A1 (described above) in the presence of human DNA. First primer “RLX0892” (described above) and second primer “RLX0893” (described above) were used to amplify T124A1.

160 microliter reaction mixtures were prepared, each containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 20 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 0.4×SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/murine RNase inhibitor (New England Biolabs), 0.8 μM of first primer RLX0892, 0.8 μM of second primer RLX0893, and 1,000, 100 or 0 copies T124A1 template per microliter, and 0, 1, 2.5, 5, or 10 nanograms human genomic DNA, and incubated at 56 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). The inflection points for the assays are shown in FIG. 5. The X-axis provides the quantity of human genomic DNA (“hDNA”) in the reaction, and the Y-axis provides the inflection time (in minutes) of the assay. For each quantity of hDNA, 3 adjacent bars are shown, from left to right: 1000 copies template/microliter, 100 copies template/microliter, or no template. As shown in FIG. 5, under these reaction conditions, the assays effectively amplified the template at 1000 copies template/microliter in the presence of at least 10 ng hDNA. Also, the addition of hDNA the reactions caused only a relatively small decrease in NTC inflection times, even at concentrations of 5 or 10 ng hDNA.

Example 5—Amplification of a Template Nucleic Acid from a Viral Lysate Eluate

A method as provided herein was used to amplify a target nucleic acid from two different samples containing the target. Target nucleic acid T124A1 was prepared in two samples: 1) a sample containing isolated T124A1 RNA molecule (as in Example 1), and 2) a sample containing isolated nucleic acids from an influenza A H3N2 viral sample. T124A1 is a portion of the HA3 gene, and therefore is expected to be present in a sample of isolated nucleic acids from influenza A H3N2 virus. Influenza A viral lysate was prepared with a Chemagic viral DNA/RNA kit (PerkinElmer), according to the manufacturer's instructions. The concentration of T124A1 in the two different samples was quantified by qPCR, and the samples were diluted to normalize the concentration of T124A1 in the samples. First primer “RLX0892” and second primer “RLX0893” were used to amplify T124A1.

25 microliter reaction mixtures were prepared, each containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 20 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 2 uM SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/μl murine RNase inhibitor (New England Biolabs), 0.8 μM of first primer RLX0892, 0.8 μM of second primer RLX0893, and 10,000, 1000 or 0 copies T124A1 template per microliter, and incubated at 56 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). The inflection points for the assays are shown in FIG. 6. The X-axis provides the copies of template/microliter in the reaction, and the Y-axis provides the inflection time (in minutes) of the assay. For each quantity of copies of template/microliter, 2 adjacent bars are shown, from left to right: isolated T124A1, and influenza A viral lysate. The inflection point for the NTC is also shown. As shown in FIG. 6, under these reaction conditions, the assays effectively amplified both isolated T124A1 template, as well as T124A1 template in an influenza A viral lysate.

Example 6—Amplification of a Template Nucleic Acid

A method as provided herein was used to amplify a target nucleic acid. Reactions were prepared to assay for a 54 nucleotide portion of T129D1, which is a 298 nucleotide portion of an influenza B virus hemagglutinin (HA) gene (an RNA molecule). The nucleotide sequence of T129D1 is provided in SEQ ID NO: 90. First primer “A8” (nucleotide sequence: 5′ TCTTGAGAGAACCCACTAAC 3′) (SEQ ID NO: 86) and second primer “B8” (nucleotide sequence: 5′ TCTCAAGAATTTGGTCTTCC 3′) (SEQ ID NO: 87) were used to amplify T129D1. In both of these primers, the first 8 nucleotides (from the 5′ terminus) are the tail region, and the last 12 nucleotides are the template-binding region. Together, these primers target a 54 nucleotide portion of T129D1.

A 100 microliter reaction mixture was prepared, containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 20 μg bovine serum albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, 0.4×SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.016 units/μl AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/μl murine RNase inhibitor (New England Biolabs), 0.8 μM of first primer A8, 0.8 μM of second primer B8, and 100,000 T129D1 template per microliter, and incubated at 58 C for 100 minutes in a CFX 96 Touch instrument (Bio-Rad). After the 100 minutes, a sample was taken from the reaction, and ligated into cloning vectors. Vectors containing reaction products were sequenced using vector-specific primers, directed to toward the cloning site. A portion of the results from multiple example sequencing reactions are shown in FIG. 7. The sequencing results show that concatemers having the expected structure are formed. Specifically, in these examples, in a single strand of the reaction product, multiple copies of the targeted 54 nucleotide sequence from the T129D1 gene are present, separated by the sequence of the tail region of the A8 primer (5′ TCTTGAGA 3′) (SEQ ID NO: 88). In FIG. 7, only a portion of the second (from left to right) occurrence of the T129D1 is shown; the sequences continue beyond the nucleotides shown in the figure.

Example 7—Primers with Tails Complementary to an Internal Motif

To further describe embodiments provided herein in which a primer tail contains a sequence which is complementary to an internal motif within a target nucleic acid, an example with exemplary nucleic acid sequences will now be provided. A polynucleotide template of 54 nucleotides total length may be provided (“Example 7 Template”). Example 7 Template corresponds to element 1000 of FIG. 10. The sequence of the Example 7 Template is as follows:

(SEQ ID NO: 92) 5′ TCGCTTGAACTGAGGACTTAGCGCCTAGAAACTTGGCTCAGGTAACC AACTGGT 3′. Describing the Example 7 Template, in the 5′ to the 3′ direction, the first underlined sequence is the second portion of the polynucleotide template (e.g. similar to element 1002 as provided in FIG. 10), the second underlined sequence is the internal motif (e.g. corresponding to element 1003 provided in FIG. 10), and the third underlined sequence is the first portion of the polynucleotide template (e.g. corresponding to element 1001 provided in FIG. 10). Specific nucleotides of the Example 7 Template and other sequences provided herein may be referred to in regards to their position in the respective sequence read in the 5′ to 3′ direction. Thus, for example, the “T” at the 5′ end of the Example 7 Template is in the 1^(st) position, the “C” adjacent to the “T” at the 5′ end is in the 2^(nd) position, and so on. A first primer of 24 nucleotides total length may be provided (“Example 7 First Primer”). Example 7 First Primer corresponds to element 1010 of FIG. 10. The sequence of the Example 7 First Primer is as follows:

(SEQ ID NO: 93) 5′ CTAGAAACACCAGTTGGTTACCTG 3′. The underlined sequence is the first/tail region of the Example 7 First Primer (corresponding to element 1011 of FIG. 10), and the non-underlined sequence is the second/template-binding region of the Example 7 First Primer (corresponding to element 1012 of FIG. 10). Also of note is the fact that the first region of the Example 7 First Primer has the same nucleotide sequence as the internal motif of the Example 7 Template. Example 7 First Primer can anneal to the Example 7 Template as follows: When the Example 7 Template and Example 7 First Primer are oriented so that the 3′ end of the Example 7 Template and the 5′ end of the Example 7 First Primer face the same direction, the non-underlined sequence of the Example 7 First Primer (read in the 5′ to 3′ direction, as in ACCAG . . . ) is annealed to the first portion of the Example 7 Template (read in the 3′ to 5′ direction, as in TGGTC . . . ). More specifically, the “A” in the 9^(th) position of the Example 7 First Primer pairs with the “T” in the 54^(th) position of the Example 7 Template, the “C” in the 10^(th) position of the Example 7 First Primer pairs with the “G” in the 53^(rd) position of the Example 7 Template, the “C” in the 11^(th) position of the Example 7 First Primer pairs with the “G” in the 52^(nd) position of the Example 7 Template, etc. When the Example 7 First Primer is annealed to the Example 7 Template, a polymerase having strand displacement activity may extend the Example 7 First Primer, as described elsewhere herein, to generate an Example 7 First Primer Extension Product. The Example 7 First Primer Extension Product will have the nucleotide sequence in the 5′ to 3′ direction of:

(SEQ ID NO: 94) 5′ CTAGAAACACCAGTTGGTTACCTGAGCCAAGTTTCTAGGCGCTAAGT CCTCAGTTCAAGCGA 3′. Describing the Example 7 First Primer Extension Product, in the 5′ to 3′ direction, the first non-underlined sequence is the sequence of the first region of the Example 7 First Primer, the first underlined sequence is a sequence which is complementary to the first portion of the Example 7 Template, the second underlined sequence is the sequence of the internal motif of the Example 7 First Primer Extension Product, and the third underlined sequence is a sequence which is complementary to the second portion of the Example 7 Template. In embodiments, when the Example 7 First Primer Extension Product is annealed to the Example 7 Template, in some circumstances, the portion of the Example 7 First Primer Extension Product which is the sequence of the first region of the Example 7 First Primer may temporarily anneal to the internal motif of the Example 7 First Primer Extension Product, such that the “C” in the 1st position anneals to the “G” in the 38^(th) position of the Example 7 First Primer Extension Product, the “T” in the 2^(nd) position anneals to the “A” in the 37^(th) position, the “A” in the 3^(rd) position anneals to the “T” in the 36^(th) position, etc., up to the “C” in the 8^(th) position annealing to the “G” in the 31^(st) position. When the portion of the Example 7 First Primer Extension Product which is the sequence of the first region of the Example 7 First Primer temporarily anneals to the portion of the internal motif of the Example 7 First Primer Extension Product, the portion of the Example 7 First Primer Extension Product between these portions (including, for example, the sequence which is complementary to the first portion of the Example 7 Template) may become un-annealed from the Example 7 Template, thus facilitating, for example, the binding of another copy of the Example 7 First Primer to the Example 7 Template, and the subsequent generation of additional Example 7 First Primer Extension Products.

A second primer of 24 nucleotides total length may also be provided (“Example 7 Second Primer”). Example 7 Second Primer corresponds to element 1020 of FIG. 10. The sequence of the Example 7 Second Primer is as follows: 5′ GTTTCTAGTCGCTTGAACTGAGGA 3′ (SEQ ID NO: 95). The underlined sequence is the first region of the Example 7 Second Primer (corresponding to element 1021 of FIG. 10), and the non-underlined sequence is the second region of the Example 7 Second Primer (corresponding to element 1022 of FIG. 10). The Example 7 Second Primer may anneal to the Example 7 First Primer Extension Product as follows: The “T” in the 9^(th) position of the Example 7 Second Primer pairs with the “A” in the 62^(nd) position of the Example 7 First Primer Extension Product, the “C” in the 10^(th) position of the Example 7 Second Primer pairs with the “G” in the 61^(st) position of the Example 7 First Primer Extension Product, the “G” in the 11^(th) position of the Example 7 Second Primer pairs with the “C” in the 60^(th) position of the Example 7 First Primer Extension Product, etc. The Example 7 Second Primer may be extended by a polymerase having strand displacement activity as described elsewhere herein, to yield an Example 7 Second Primer Extension Product. The Example 7 Second Primer Extension Product will have the nucleotide sequence in the 5′ to 3′ direction of:

(SEQ ID NO: 96) 5′ GTTTCTAGTCGCTTGAACTGAGGACTTAGCGCCTAGAAACTTGGCTC AGGTAACCAACTGGTGTTTCTAG 3′. Describing the Example 7 Second Primer Extension Product, in the 5′ to 3′ direction, the first non-underlined sequence is the sequence of the first region of the Example 7 Second Primer, the first underlined sequence the sequence of the second portion of the Example 7 Template, the second underlined sequence is the sequence of the internal motif of the Example 7 Second Primer Extension Product, the third underlined sequence is the sequence of the first portion of the Example 7 Template, and the fourth non-underlined sequence is the sequence of the first region of the Example 7 Second Primer and the sequence of a sequence which is complementary to the first region of the Example 7 First Primer. In embodiments, when the Example 7 Second Primer Extension Product is annealed to the Example 7 First Primer Extension Product, in some circumstances, the portion of the Example 7 Second Primer Extension Product which is the sequence of the first region of the Example 7 Second Primer may temporarily anneal to the internal motif of the Example 7 Second Primer Extension Product, such that the “G” in the 1^(st) position anneals to the “C” in the 40^(th) position of the Example 7 Second Primer Extension Product, the “T” in the 2^(nd) position anneals to the “A” in the 39^(th) position, the “T” in the 3^(rd) position anneals to the “A” in the 38^(th) position, etc., up to the “G” in the 8^(th) position annealing to the “C” in the 33^(rd) position. When the portion of the Example 7 Second Primer Extension Product which is the sequence of the first region of the Example 7 Second Primer temporarily anneals to the internal motif of the Example 7 Second Primer Extension Product, the portion of the Example 7 Second Primer Extension Product between these portions (including, for example, the sequence of the second portion of the Example 7 Template) may become un-annealed from the Example 7 First Primer Extension Product, thus facilitating, for example, the binding of another copy of the Example 7 Second Primer to the Example 7 First Primer Extension, and the subsequent generation of additional Example 7 Second Primer Extension Products.

In embodiments, methods provided elsewhere herein may proceed according to the general mechanisms outlined above. Any of the descriptions and details provided elsewhere herein for primers, target nucleic acids, reaction conditions, reaction products, etc. may apply to reactions involving the general mechanism described above in Example 7.

Example 8—SNP Detection

A method provided herein was used for identifying a SNP/mutation in a target nucleic acid. Two versions of the target nucleic acid were provided, which differed by a single nucleotide. The first strand of Version 1 of the nucleic acid template (“Version 1 Template”; also referred to as the “wild-type” sequence) has the sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 97) GCATTCATCACGTTTTTGCGAGTCCTTTCC

TCCCACCAACAGCAGGGAT TCTGAAGAGATGGGGACAGTT. Describing the Version 1 Template, in the 5′ to 3′ direction, the first underlined sequence is the second portion of the Version 1 Template, the second underlined sequence is the internal motif of the Version 1 Template, and the third underlined sequence is the first portion of the Version 1 Template. The first strand of Version 2 of the nucleic acid template (“Version 2 Template”; also referred to as the “SNP” sequence) has the sequence in the 5′ to 3′ direction:

(SEQ ID NO: 98) GCATTCATCACGTTTTTGCGAGTCCTTTCC

TCCCACCAACAGCAGGGAT TCTGAAGAGATGGGGACAGTT. Describing the Version 2 Template, in the 5′ to 3′ direction, the first underlined sequence is the second portion of the Version 2 Template, the second underlined sequence is the internal motif of the Version 2 Template, and the third underlined sequence is the first portion of the Version 2 Template. The Version 1 Template and the Version 2 Template sequences differ only in a single nucleotide in the internal motif sequence of the respective Templates. Specifically, the Version 1 Template has an “A” in the 31^(st) position in the sequence (emphasized with bold and italicized font), whereas the Version 2 Template has a “T” in the 31^(st) position in the sequence (emphasized with bold and italicized font). A first primer and a second primer were also provided to amplify the Template sequences. The first primer (“Example 8 Primer 1”) has a sequence of:

(SEQ ID NO: 99) 5′ TCCATCCCAACTGTCCCCATCT 3′. The underlined sequence is the first/tail region of the Example 8 Primer 1, and the non-underlined sequence is the second/template-binding region of the Example 8 Primer 1. The second primer (“Example 8 Primer 2”) has a sequence of:

(SEQ ID NO: 100) 5′ GGGATGGAGCATTCATCACGTTT 3′. The underlined sequence is the first/tail region of the Example 8 Primer 2, and the non-underlined sequence is the second/template-binding region of the Example 8 Primer 2. Separate amplification reactions as described elsewhere herein were performed for either the Version 1 Template or the Version 2 Template, with the reactions for both the Version 1 Template and the Version 2 Template using Example 8 Primer 1 and Example 8 Primer 2 as the primers. Multiple reactions under the same conditions were performed for each Template.

In the Version 1 Template reactions, upon the annealing of the Example 8 Primer 1 to the Version 1 Template and the generation by a polymerase of an extension product of the Example 8 Primer 1 (“Version 1 Template Primer 1 Extension Product”), the Version 1 Template Primer 1 Extension Product will have a sequence as follows: 5′

(SEQ ID NO: 101)  TCCATCCCAACTGTCCCCATCTCTTCAGAATCCCTGCTGTTGGTGGGA

G GAAAGGACTCGCAAAAACGTGATGAATGC. Describing the Version 1 Template Primer 1 Extension Product, in the 5′ to 3′ direction, the first non-underlined sequence is the sequence of the first/tail region of the Example 8 Primer 1, the first underlined sequence is a sequence which is complementary to the first portion of the Version 1 Template, the second underlined sequence is the sequence of the internal motif of the Version 1 Template Primer 1 Extension Product (where the bold, italicized “T” is the nucleotide of interest), and the third underlined sequence is a sequence which is complementary to the second portion of the Version 1 Template. According to methods described herein, in embodiments, the portion of the Version 1 Template Primer 1 Extension Product corresponding to the sequence of the first/tail region of the Example 8 Primer 1 sequence may temporarily anneal to the internal motif of the Version 1 Template Primer 1 Extension Product, such that the “T” in the 1^(st) position anneals to the “A” in the 52^(nd) position, the “C” in the 2^(nd) position anneals to the “G” in the 51^(st) position, the “C” in the 3^(rd) position anneals to the “G” in the 50^(th) position, etc., up to the “C” in the 8^(th) position annealing to the “G” in the 45^(th) position. Of note, the portion of the Version 1 Template Primer 1 Extension Product corresponding to the sequence of the first/tail region of the Example 8 Primer 1 sequence is perfectly complementary to the internal motif sequence of the Version 1 Template Primer 1 Extension Product (i.e. there are no mis-matches between any of the 8 sets of paired nucleotides).

In the Version 2 Template reactions, upon the annealing of the Example 8 Primer 1 to the Version 2 Template and the generation by a polymerase of an extension product of the Example 8 Primer 1 (“Version 2 Template Primer 1 Extension Product”), the Version 2 Template Primer 1 Extension Product will have a sequence as follows:

(SEQ ID NO: 102) 5′ TCCATCCCAACTGTCCCCATCTCTTCAGAATCCCTGCTGTT GGTGGGA

GGAAAGGACTCGCAAAAACGTGATGAATGC. Describing the Version 2 Template Extension Product, in the 5′ to 3′ direction, the first non-underlined sequence is the sequence of the first/tail region of the Example 8 Primer 1, the first underlined sequence is a sequence which is complementary to the first portion of the Version 2 Template, the second underlined sequence is the sequence of the internal motif of the Version 2 Template Primer 1 Extension Product (where the bold, italicized “A” is the nucleotide of interest), and the third underlined sequence is a sequence which is complementary to the second portion of the Version 2 Template. According to methods described herein, in embodiments, the portion of the Version 2 Template Primer 1 Extension Product corresponding to the sequence of the first/tail region of the Example 8 Primer 1 sequence may temporarily anneal to the internal motif of the Version 2 Template Primer 1 Extension Product, such that the “T” in the 1^(st) position anneals to the “A” in the 52^(nd) position, the “C” in the 2^(nd) position anneals to the “G” in the 51^(st) position, the “C” in the 3^(rd) position anneals to the “G” in the 50^(th) position, etc., up to the “C” in the 8^(th) position annealing to the “G” in the 45^(th) position. Of note, the portion of the Version 2 Template Primer 1 Extension Product corresponding to the sequence of the first/tail region of the Example 8 Primer 1 sequence is not perfectly complementary to the internal motif sequence of the Version 2 Template Primer 1 Extension Product, as there is a mis-match between the “A” in the 4^(th) position and the “A” in the 49^(th) position. Thus, comparing the Version 1 Template Primer 1 Extension Product to the Version 2 Template Primer 1 Extension Product, the portion of the respective Extension Product corresponding to the first/tail region of the Example 8 Primer 1 will have greater complementation to and will more stably anneal to the internal motif in the Version 1 Template Primer 1 Extension Product than in Version 2 Template Primer 1 Extension Product. In particular, the sequence “TCCATCCC” in the Version 1 Template Primer 1 Extension Product is perfectly complementary to the internal motif in the Version 1 Template Primer 1 Extension Product (8 out of 8 nucleotides can properly pair), whereas the sequence “TCCATCCC” in the Version 2 Template Primer 1 Extension Product is not perfectly complementary to the internal motif in the Version 2 Template Primer 1 Extension Product (only 7 out of 8 nucleotides can properly pair). Typically, greater complementarity between the portion of an Extension Product corresponding to a primer tail and an internal motif in the Extension Product results in a more stable annealing between these two portions. When an Extension Product is configured such that these two portions are annealed, it may accelerate the rate of a reaction provided herein, for example, by permitting the binding of a new primer to un-annealed nucleotides in a complementary strand exposed by the internal annealing of regions of an Extension Product or by the increased rate of concatemer formation according to methods provided herein.

A similar mechanism as described above also occurs for the Version 1 Template and Version 2 Template Primer 2 Extension Products, where the portion of the tail region of the Extension Products is perfectly complementary for the Version 1 Template Primer 2 Extension Product, but is not perfectly complementary for the Version 2 Template Primer 2 Extension Product.

FIG. 11 shows the results of the amplification reactions with these templates and primers, where the X-axis shows time and the Y-axis shows relative fluorescence units (RFU) (indicating amplified DNA). As shown in FIG. 11, the amplification reactions with the Version 1 Template (“wild-type” sequence) occurred more quickly than reactions with the Version 2 Template (“SNP” sequence). Thus, these results show that methods provided herein can be used, for example, to distinguish between sequences having single-nucleotide variations.

Example 9—SNP Detection

A method provided herein was used for identifying a SNP/mutation in a human target nucleic acid. The Q356R variant of the BRCA1 protein may be associated with an increased risk of cancer. The Q356R variant may occur, for example, when the codon (nucleotide sequence CAG) encoding the glutamine at the 356 amino acid position of the wild-type BRCA1 protein instead has the nucleotide sequence CGG, so that the codon encodes an arginine instead of glutamine.

Four different versions of a portion of the BRCA1 genomic sequence which contains, among other nucleotides, the codon for the 356 amino acid position were obtained. The first version (“Wild-Type Target”) contains the wild-type codon encoding the glutamine at the 356 amino acid position (sequence CAG), and has the nucleotide sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 103) AAGATGTTCCTTGGATAACACTAAATAGCAGCATTC

GAAAGTTAATGAG TGGTTTTCCAGA. The second version (“Q356R Target”) contains a codon encoding an arginine at the 356 amino acid position (sequence CGG), and has the nucleotide sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 104) AAGATGTTCCTTGGATAACACTAAATAGCAGCATTC

GAAAGTTAATGAG TGGTTTTCCAGA. The third version (“Q356P Target”) contains a codon encoding a proline at the 356 amino acid position (sequence CCG), and has the nucleotide sequence in the 5′ to 3′ direction:

(SEQ ID NO: 105) AAGATGTTCCTTGGATAACACTAAATAGCAGCATTC

GAAAGTTAATGAG TGGTTTTCCAGA.

The fourth version (“Q356L Target”) contains a codon encoding a leucine at the 356 amino acid position (sequence CTG), and has the nucleotide sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 106) AAGATGTTCCTTGGATAACACTAAATAGCAGCATTC

GAAAGTTAATGAG TGGTTTTCCAGA.

In each of the above four Targets, in the 5′ to 3′ direction, the first underlined sequence is the second portion of the Target, the second underlined sequence is the internal motif of the Target, and the third underlined sequence is the first portion of the Target.

The above four Target sequences differ by only a single nucleotide in the internal motif sequence of the respective Targets. Specifically, the Wild-Type Target has an “A” in the 37^(th) position in the sequence (emphasized with bold and italicized font), whereas the Q356R Target has a “G” in the 37^(st) position in the sequence, the Q356P Target has a “C” in the 37^(st) position in the sequence, and the Q356L Target has a “T” in the 37^(st) position in the sequence. A first primer and a second primer were also provided to amplify the Target sequences. The first primer (“Example 9 Primer 1”) has a sequence of:

(SEQ ID NO: 107) 5′ ATTCAGAATCTGGAAAACCACTC 3′. The underlined sequence is the first/tail region of the Example 9 Primer 1, and the non-underlined sequence is the second/template-binding region of the Example 9 Primer 1. The second primer (“Example 9 Primer 2”) has a sequence of:

(SEQ ID NO: 108) 5′ TTCTGAATAAGATGTTCCTTGGAT 3′.

The underlined sequence is the first/tail region of the Example 9 Primer 2, and the non-underlined sequence is the second/template-binding region of the Example 9 Primer 2. Separate amplification reactions as described herein were performed for each of the above four Targets, with the reactions for all four Targets using Example 9 Primer 1 and Example 9 Primer 2 as the primers. The conditions for the reactions were as follows: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT, 0.8 M betaine, 2 uM SYTO® 59 (Life Technologies), 0.8 units/μl Bst DNA polymerase (New England BioLabs), 0.8 μM of Example 9 Primer 1, 0.8 μM of Example 9 Primer 2, and 10 copies Target molecule/μl. The reactions were held at 56 C for 90 minutes, and the inflection point of each reaction determined using a single-threshold method with CFX Manager software (Bio-Rad). Multiple reactions under the same conditions were performed for each Target.

The amplification reactions occurred by mechanisms described elsewhere herein, for example, as in Example 8. For example, Example 9 Primer 1 and Example 9 Primer 2 extension products were formed from each of the above Target nucleic acids, in each respective reaction, to form, for example a Wild-Type Target Example 9 Primer 1 Extension Product, a Q356R Target Example 9 Primer 1 Extension Product, a Q356P Target Example 9 Primer 1 Extension Product, and a Q356L Target Example 9 Primer 1 Extension Product, and corresponding extension products from each target from Example 9 Primer 2.

Of note, whereas the portion of, for example, the Wild-Type Target Example 9 Primer 1 Extension Product corresponding to the first/tail region of the Example 9 Primer 1 is perfectly complementary to the internal motif in the Wild-Type Target Example 9 Primer 1 Extension Product, the portion of the Extension Product corresponding to the first/tail region of the Example 9 Primer 1 for each of the Q356R Target, Q356P Target, and Q356L Target Primer 1 Extension Products is not perfectly complementary to the internal motif in the respective Extension Product. Specifically, there is a single-nucleotide mis-match at the position involving the nucleotide in the 37^(th) position of the target sequence, as described above. This mismatch may affect the rate of a reaction provided herein.

The average inflection time of the assays with each of the different Targets was as follows: Wild-Type Target: 24.5 minutes; Q356R Target: 49.5 minutes, Q356P Target: 55.7 minutes, and Q356L Target: 59.8 minutes. Thus, as indicated by the inflection times of the assays, the reactions can readily differentiate between targets which vary by a single nucleotide in an internal motif. The significantly faster inflection time of the assay with the Wild-Type Target as compared to the other Targets may be a result of, for example, the perfect complementation between the portion of the Wild-Type Target Example 9 Primer 1 Extension Product corresponding to the first/tail region of the Example 9 Primer 1 and the internal motif in the Wild-Type Target Example 9 Primer 1 Extension Product, in contrast to the imperfect complementation between the portion of the Q356R Target, Q356P Target, and Q356L Target Example 9 Primer 1 Extension Product which corresponds to the first/tail region of the Example 9 Primer 1 and the internal motif in the respective Example 9 Primer 1 Extension Product. A similar reasoning also applies to the extension products of the Primer 2 Extension Products.

Example 10—Influenza N9 Gene Amplification

A method provided herein was used to amplify a portion of the neuraminidase gene (N9) from a sample containing Influenza A virus, subtype H7N9. The target sequence from N9 was, in the 5′ to 3′ direction (in DNA form):

(SEQ ID NO: 109) AACTTTAAGGACGAACAAGAGAGAAGGTAAACCTACAACCACTACAAACA AAGTAATATTATCAACAAATAATAACAAACCGAACACAACCAACAAAGT. The first primer used to amplify the template had the sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 110) CTTCCATTTGAAACAACCAACAC. Describing the first primer, the underlined region is the tail/first region of the primer and the non-underlined region is the second/template-binding region. The second primer used to amplify the template had the sequence, in the 5′ to 3′ direction:

(SEQ ID NO: 111) AATGGAAGTTGAAATTCCTGCTTG. Describing the first primer, the underlined region is the tail/first region of the primer and the non-underlined region is the second/template-binding region. Multiple reactions as described herein were performed using the first primer and second primer, where the reactions either contained template sequence or did not contain template sequence. The reactions containing the template had 250,000 copies template sequence per reaction. The results of the reactions are provided in FIG. 12. The numbers on the X-axis are minutes, and the Y-axis values are relative fluorescence units (RFU). As shown in the graph, in the templated reactions, the RFU rapidly rises at about 20 minutes, indicating the amplification of the template sequence. The increase in RFU in the no template reactions at about 60 minutes and later is a result of non-specific background products.

Nucleotide and amino acid sequences provided herein are artificial sequences, unless otherwise noted.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The foregoing description is not intended to be exhaustive or to limit the invention to the precise embodiments disclosed, and other modifications and variations may be possible in light of the above teachings without departing from the invention. Any feature, whether preferred or not, may be combined with any other feature, whether preferred or not. It should also be understood that while the invention provided herein has been described herein using a limited number of terms and phrases for purposes of expediency, the invention could also be described using other terms and phrases not provided herein which also accurately describe the invention. The appended claims are not to be interpreted as including means-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase “means for.” It should be understood that as used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. For example, a reference to “an assay” may refer to a single assay or multiple assays. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. As used in the description herein and through the claims that follow, a first object described as containing “at least a portion” of a second object may contain the full amount of/the complete second object. As used in the description herein and throughout the claims that follow, the terms “comprise”, “include”, and “contain” and related tenses are inclusive and open-ended, and do not exclude additional, unrecited elements or method steps. Also, the presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. Finally, as used in the description herein and throughout the claims that follow, the meaning of “or” includes both the conjunctive and disjunctive unless the context expressly dictates otherwise. Thus, the term “or” includes “and/or” unless the context expressly dictates otherwise.

This document contains material subject to copyright protection. The copyright owner (Applicant herein) has no objection to facsimile reproduction by anyone of the patent documents or the patent disclosure, as they appear in the US Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. The following notice shall apply: Copyright 2013-15 Theranos, Inc. 

1-81. (canceled)
 82. A reaction mixture comprising: a polynucleotide template, a first primer, and a second primer, wherein: the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion; the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to the second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and the first region of the second primer is complementary to the third portion of the polynucleotide template.
 83. The reaction mixture of claim 82, wherein the reaction mixture further comprises a DNA polymerase having strand-displacement activity.
 84. The reaction mixture of claim 82, wherein the reaction mixture further comprises a reverse transcriptase.
 85. The reaction mixture of claim 82, wherein the reaction mixture further comprises a nucleic acid dye.
 86. The reaction mixture of claim 82, wherein the reaction mixture further comprises nucleotides and buffer.
 87. The reaction mixture of claim 82, wherein the polynucleotide template is a DNA strand.
 88. The reaction mixture of claim 82, wherein the polynucleotide template is an RNA strand.
 89. The reaction mixture of claim 82, wherein the polynucleotide template is part of a duplex DNA molecule.
 90. The reaction mixture of claim 82, wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length.
 91. The reaction mixture of claim 82, wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length.
 92. A kit for the amplification of a polynucleotide template, the kit comprising: a first primer and a second primer, wherein: the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of the polynucleotide template; the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the first primer is complementary to the first region of the second primer; and the first region of the second primer is complementary to a third portion of the polynucleotide template, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion.
 93. (canceled)
 94. The kit of claim 92, further comprising: a third primer, and a fourth primer, wherein: the third primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template; the fourth primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template; the first region of the third primer is complementary to the first region of the fourth primer; and the nucleotide sequence of the first region of the first primer differs from the nucleotide sequence of first region of the third primer by a single nucleotide.
 95. The kit of claim 94, wherein the first region of the fourth primer is complementary to a third portion of the polynucleotide template, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion. 96-102. (canceled)
 103. The method of claim 69, wherein the polynucleotide template is a DNA strand.
 104. The method of claim 69, wherein the polynucleotide template is an RNA strand.
 105. The method of claim 69, wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length.
 106. The method of claim 69, wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length.
 107. The method of claim 69, wherein the number of copies of the polynucleotide template in the reaction mixture is increased at least 10-fold within 60 minutes of initiation of the method.
 108. The method of claim 78, wherein the reaction mixture is incubated without thermocycling. 